Objective To explore the intervention measures and effectiveness evaluation for the improvement of the pathogenic detection rate related to healthcare-associated infection (HAI) diagnosis in secondary general hospitals. Methods The reported HAI cases among all hospitalized patients in a secondary general hospital from 2021 to 2022 were selected as the research object, with 2021 as the pre-intervention group and 2022 as the post-intervention group. Indicators of pathogenic detection rates related to HAI diagnosis before and after the implementation of improvement intervention measures were compared. Results Pathogenic detection rate related to HAI diagnosis was 66.07% in 2021. After implementing improvement intervention measures, detection rate reached 91.55% in 2022, meeting the standard requirements of ≥90%. The difference before and after intervention was statistically significant (χ²=12.85, P < 0.01). After implementing improvement intervention measures, differences in the antimicrobial use rate and the constituent ratios of restricted and special-grade antimicrobials among hospitalized patients were all statistically significant (all P < 0.05). Conclusion Implementing effective improvement intervention measures can increase HAI diagnosis-related pathogenic detection rate, but the quality control indicators still needs to be improved and refined.