Objective To evaluate the impact of intensified infection control measures on the incidence of healthcare-associated infection (HAI) under the background of the reform of diagnosis-intervention packet (DIP) payment, and provide decision-making basis for HAI management under the reform of medical insurance payment. Methods The interrupted time series research design was used to collect the monitoring data of HAI in a tertiary first-class hospital from October 2021 to September 2024. The changing trend of HAI incidence was analyzed by piecewise li-near regression, and the intervention effect was evaluated by standardized infection ratio (SIR). Results From October 2021 to September 2022, the incidence of HAI was 2.17% (95%CI: 2.08%-2.26%), which dropped to 1.87% (95%CI: 1.82%-1.92%) after the intervention. Piecewise linear regression analysis showed that the incidence of HAI decreased by 0.324% immediately after the intervention (95%CI: -0.481%- -0.167%, P<0.001), and the trend after the intervention changed significantly compared with that before the intervention (95%CI: -0.033%- -0.009%, P=0.001). SIR analysis showed that the actual incidence during the entire intervention period was equivalent to 74.56% of the incidence in intervention period, gradually stabilized from 88.39%-93.81% at the beginning of the intervention to 67.03%-71.22% at the end of the study, and the intervention effect was sustained. Conclusion Intensified infection control measures under the background of the reform of DIP significantly reduce the incidence of HAI and improve the stability of infection control management, which provide new insights into the synergistic improvement of medical insurance payment and HAI management quality.