Objective To analyze the changing trend of the incidence of healthcare-associated infection (HAI) before and after the response of medical institutions to major public health emergencies, and provide a basis for the construction of emergency response system of infection prevention and control (IPC) in medical institutions under major public health emergencies. Methods HAI incidence density rate in a tertiary first-class specialty hospital from January 2018 to June 2021 were analyzed. Enhanced IPC measures in response to public health emergencies on the incidence of HAI were quantitatively evaluated with piecewise regression of interrupted time-series (ITS) analysis. Results From 2018 to 2019, the baseline HAI incidence density rate of this hospital before the intervention with enhanced IPC measures was 1.44 ‰(95%CI: 1.35‰-1.53‰). From January 2020 to June 2021, HAI incidence density rate after the intervention was 0.94‰ (95%CI: 0.88‰-1.02‰). In December 2019, HAI incidence density rate immediately dropped after the launch of the enhanced IPC measures, with significant short-term effect(aIRR=57.60%, 95%CI: 46.84%-70.66%, P < 0.001). HAI incidence density rate gradually increased thereafter, with a long-term slope of 100.08% (aIRR=100.08%, 95%CI: 100.04%-100.12%, P < 0.001). Stan-dardized infection ratio (SIR) of the actual number of emerging HAI per month after intervention to the number from model prediction without intervention showed that except January 2020 (SIR: 0.62, 95%CI: 0.41-0.91, P=0.013), February 2020 (SIR: 0.51, 95%CI: 0.31-0.78, P < 0.001), March 2020 (SIR: 0.37, 95%CI: 0.21-0.61, P < 0.001), and July 2020 (SIR: 0.48, 95%CI: 0.29-0.75, P < 0.001), HAI incidence density rate in other months after intervention were not significantly different from that before intervention. Conclusion The enhanced IPC measures in response to major public health emergencies can significantly reduce HAI incidence density rate of each month in a short period, but the effect gradually disappears over time and returns to the pre-intervention level. The specific causes for the V-shaped effect of intervention need to be further analyzed.