Objective The difference-in-differences (DID) estimation was used to accurately evaluate the effect of anti-parasitic disease intervention on children’s health-related behaviors and soil-transmitted nematode infection. Methods Children in Yi villages in Liangshan Yi Autonomous Prefecture, Sichuan Province, were selected based on a multistage stratified random cluster sampling method for the investigation in 2016. The demographic information of the participated children and their health-related behaviors were collected by a issued questionnaire. Fecal samples were collected from participated children with age ≥ 3 and the eggs of soil-transmitted nematodes were examined using Kato-Katz method under microscope. The children were randomly divided into the intervention and control group in the same village. A comprehensive intervention measures, including health education and anthelmintic treatment, were performed in the intervention group. One year after the intervention, the same method was used to collect the relevant information from the follow-upped children. Using the DID model, the accurate effect of the intervention on children’s health-related behaviors and the control of soil-transmitted nematode infection was analyzed. Results Total 162 children were recruited in this study with an average age of 7.7 years old. They were divided into the intervention group (99) and the control group (63). After the intervention, the DID analysis results demonstrated that the sanitation and hygiene behaviors have been significantly improved. These behaviors included the hand washing before meal at home (DID = 37.5%, P < 0.05), hand washing before meal in school (DID = 24.4%, P < 0.01), hand washing after toilet at home (DID = 45.3%, P < 0.01), hand washing after toilet in school (DID = 35.9%, P < 0.01) and brushing teeth and washing face (DID = 29.6%, P < 0.01). The positive effect was also reflected by the reduced infection rate of Ascaris lumbricoides (DID = -37.5%, P < 0.01). However, other DID values for some high-risk behaviors (eating unwashed fruits and vegetables, drinking un-boiled water, walking in barefoot, playing with mud on the ground and sucking fingers) and infection rates of hookworm and Trichuris trichiura were not significantly improved (P > 0.05). Conclusion After the interventions, some hygiene behaviors, as well as A. lumbricoides infection rate of school-age children has significantly improved. The DID estimation demonstrated the positive effect of interventions on the health-related behaviors and A. lumbricoides infection in children.