Objective To evaluate the intervention effect of comprehensive prevention and control measures on enterobiasis among children so as to provide a informative basis for formulating and adjusting the prevention and control strategies. Methods Using random cluster sampling method, one kindergarten was selected from each of the 12 districts in Nanjing to conduct a survey on the infection of Enterobius vermicularis among all children in September to October 2022. Based on the survey results, four kindergartens with higher infection rates and similar environment and scale were selected from both urban and suburban areas, respectively. All children from 8 kindergartens, one guardian living with each child and the class teacher of each class were enrolled in the survey. Among them, two kindergartens in the urban area and two in the suburbs were assigned as the experimental group implemnting comprehensive control measures, while the remaining four kindergartens as the control group carring out traditional control measures; both groups conducted one-year intervention from November 2022 to November 2023. For the experimental group, the intervention measres comprised of symptom surveillance (on children’s suspected symptoms of Enterbobius infection), companion surveillance (sampling test for positive children’s family members living together with, classmates and playmates), and environmental surveillance (using transparent adhesive tape pasting method to collect environmental samples from positive children’s families and classes), as well as health education with three-dimension of “knowledge, attitude, and practice” as entering point to perform educational intervention jointly with multiple sectors including families, schools, and communities. The intervention measures for the control group were deworming for positive children only and health education delivered to the children and their family members. The After completion of the intervention study, anal swab method with transparent adhesive tape was used to examine the E. vermicularis infection rate in the children of two groups, respectively. Before and after the inervention, questionnaire survey on E. vermicularis control related knowledge was delivered to one class teacher of each class and one guardian living with each child, respectively. Results Before the intervention, the E. vermicularis infection rates in the experimental group and the control group were 1.5% (13/885) and 1.4% (12/886), respectively, showing no significant difference between the two groups (χ² = 0.042, P > 0.05). A total of 234 children with suspected symptoms were found during symptom monitoring in the experimental group, among them, anal itching proportion was found the highes (34.62%, 81/234), followed by sleeping teeth grinding symptom (20.5%, 48/234). A total of 8 positive cases were detected, of which symptoms of anal itching accounted for the highest (5/8), followed by symptoms of sleeping teeth grinding (2/8). In companion surveillance, four positives were detected from positive children’s family members and two positives from their classmates, repectively. In environmental surveillance, four positives were found from plush toys samples, and two positive samples of blankets, sheets, and sofas were detected in the positive children’s family environment, respectively, and one positive sample was found from the desk and chair of their classroom, respectively. After one-year intervention, the infection rate in the experimental group decreased from 1.5% (13/885) before intervention to 0.1% (1/885) (χ² = 10.368, P < 0.05), which was significantly lower than that in the control group after intervention (0.9%，8/886) (χ² = 4.014, P < 0.05). The questionnaire showed that prior to the intervention, there was no significant difference in positive response rate towards children’s hygiene behaviour habits, and parents’ and teachers’ awareness points regarding to enterobiasis “knowledge, attitude, and practice” between the two the groups (all P > 0.05). The correct response rates to the questions “whether washing hands before and after meals, habits of trimming nails, biting fingers and biting toys”(89.9%, 796/885; 88.9%, 787/885; 85.8%, 759/885; 86.8%, 768/885) and correct hygiene habits rates of “whether have a separate towel, frequency of weekly showers, and frequency of monthly bedding drying” (89.2%, 789/885; 85.7%, 758/885; 78.9%, 698/885) in the experimental group were significantly higher than those in the control group (61.7%, 547/886; 71.2%, 631/886; 56.2%, 498/886; 59.8%, 530/886; 78.8%, 698/886; 78.8%, 698/886; 68.6%, 608/886) after intervention (χ² = 192.194, 86.989, 187.741, 164.402, 35.371, 14.285, 24.010; all P < 0.05). After intervention, the positive response rates to all questions towards enterobiasis “knowledge, attitude, and practice” the experimental group were significantly higher those in the control group (all P < 0.05). In post-intervention, the positive response to all questions towards awareness of enteroviasis in teachers were all significantly higher than those in the control group (all P < 0.05). Conclusion Implementation of comprehensive prevention and control measures effectively reduced E. vermicularis infection rate in children, achieved timely interruption and precise control, meanwhile, raised parents’ and teachers’ health awareness and cognitive level on enterobiasis, and promoted changes of their attitudes and behaviours, driving children to develop good hygiene habits, and leading to effectively prevent and interrupt the transmission and spread of enterobiasis.