Objective To analyze the rechecking results of the reference laboratory for malaria diagnosis in Henan Province on malaria blood samples submitted by malaria diagnosis institutions at all levels and evaluate the malaria detection capacity of malaria diagnosis institutions in Henan Province. Methods Blood smears and whole blood samples, and the diagnosis institute information, diagnosis results and other information on the malaria cases and suspected malaria cases were collected in Henan Province from 2015 to 2019. The provincial malaria reference laboratory rechecked and tested the blood samples through three methods, including microscopy, rapid diagnostic test (RDT) and PCR, and analyzed the detection results, the misjudgment of worm species, and the positive coincidence and misjudgment of worm species in diagnostic institutions at all levels. The positive coincidence rate and misdiagnosis rate of different diagnostic institutions were analyzed by χ² test. Results From 2015 to 2019, the Malaria Diagnostic Reference Laboratory of Henan Province received a total of 1 194 malaria samples, of which 1 078 samples (90.3%) were confirmed positive, and 116 negative (9.7%) by using the three testing methods. The positive coincidence rate by PCR, RDT and microscopy was 98.7% (1 064/1 078), 96.9% (1 045/1 078) and 89.1% (960/1 078), respectively. A total of 934 samples had the same results between microscopy and PCR, among them, 711 were falciparum malaria, 58 vivax malaria, 32 quartan malaria and 133 ovale malaria. From 2015 to 2019, the rate of species misidentification showed a decreasing trend, which were 12.4% (25/202), 12.2% (28/230), 7.3% (15/206), 11.3% (27/239), and 5.4% (17/317), respectively. The misdiagnosis rates of Plasmodium falciparum, P. vivax, P. ovale, P. malariae, mixed infection and P. knowlesi were 3.7% (30/820), 23.4% (15/64), 39.9% (59/148), 34.3% (12/35), 9/10 and 1/1, respectively. The malaria cases diagnosed by disease prevention and control institutions (CDC), medical institutions and other institutions accounted for 23.7% (283/1 194), 74.5% (890/1 194) and 1.8% (21/1 194) respectively. Among the 283 cases diagnosed by disease control institutions, the positive coincidence rates of provincial, municipal and county (district) level disease control institutions were 9/9, 93.2% (69/674) and 91% (182/200), respectively, with no significant difference (χ² = 0.438, P > 0.05）. The error rate of Plasmodium species was 1/9, 20.3% (14/69) and 11.5% (21/182) respectively, with no significant difference (χ² = 3.271, P < 0.05). Among 890 cases diagnosed by medical institutions, the positive coincidence rates of provincial, municipal, county (district) and township (town) medical institutions were 81.0% (17/21), 91.7% (742/809), 92.2% (47/51) and 6/9 respectively, with a statistically significant difference (χ² = 55.392, P < 0.05); the misdiagnosis rate of Plasmodium species was 7/17, 10.9% (81/742), 10.6% (5/47) and 1/6 respectively, with statistical significance (χ² = 11.978, P < 0.05). Conclusion The local settings for diagnosis units of malaria cases in Henan Province are mainly medical institutions, and the detection capacity of malaria laboratories at all levels is generally high. It is imperative to continuously strengthen. The training for malaria diagnosis professionals in medical institutions to maintain the higher capacity of malaria diagnosis.