Abstract: Objective  To analyze the implementation of HIV antibody screening at HIV-screening laboratories in Zhengzhou City for a scientific basis for further facilitating local HIV/AIDS prevention and control. Methods  Annual HIV antibody screening data from 2020 to 2024 for HIV-screening laboratories in Zhengzhou were obtained from the National AIDS Testing Laboratory Information Management System, and the number of screening tests, the number of reactive results and positivity rate were analyzed. Serum blind sample test results were obtained from the AIDS confirmation laboratory of Zhengzhou Center for Disease Control and Prevention, and the occurrence of unacceptable blind sample results was analyzed as well. Results  The number of medical and health institutions with HIV-screening laboratories in Zhengzhou increased from 106 in 2020 to 159 in 2024. Among public institutions, the number of HIV-screening laboratories was the most at general hospitals, and among non-public institutions, at specialized hospitals. Public general hospitals had the highest cumulative number of screening tests (6.349 1 million person-times), and third-party medical laboratories had the lowest (397.7 thousand person-times). The annual number of screening tests in both public and non-public specialized hospitals showed significant increasing trends (β=4.07, 0.83; t=3.35, 4.58; both P<0.05). The number of reactive results was maximal in public general hospitals (4 260 person-times), and the lowest in non-public traditional Chinese medicine hospitals (207 person-times). The highest reactive rate was observed in third-party medical laboratories (2.73‰), and the lowest in non-public traditional Chinese medicine hospitals (0.43‰). The annual trend in the reactive rate of third-party medical laboratories showed a downward trend (AAPC=-44.84%, 95% CI: -66.34% to -9.60%, t= -3.83, P<0.05). Among laboratories with unacceptable blind sample results, the annual average number and proportion were both higher in non-public institutions than in public institutions (6.8 vs. 4.4 laboratories, 11.71% vs. 6.39%). Among different types of institutions, non-public specialized hospitals had the highest annual average number and proportion (4.2 laboratories, 14.63%). Conclusion  The HIV-screening laboratory network in Zhengzhou City has been scaling up in recent years, and hospitals have played a pivotal role in case detection. Non-public institutions, especially specialized hospitals, must continuously strengthen relevant technical training and quality control to ensure the quality of testing results.