Objective To explore the effect of application of plan-do-check-action (PDCA) cycle on improving health care workers' (HCWs) compliance to hand hygiene(HH) as well as the influence on incidence of healthcare-associated infection(HAI) in patients. Methods HH questionnaire was designed and used to survey HCWs in a tertiary hospital in December 2014, bundle intervention measures were formulated according to the survey results and routine supervision problems, measures were implemented by PDCA cycle management mode from January 2015. January 2015 to December 2020 was as a post-intervention period, HH-related indicators and incidence of HAI between January-December 2014 and post-intervention period were compared. Results In December 2014, 2 513 questionnaires were distributed and 1 806 were effectively recovered, 507 doctors (28.07%) and 1 185 nurses (65.61%) were surveyed. After the continuous improvement with PDCA cycle, facility completeness rate, compliance rate, correct rate and awareness rate of HH in 2020 were 99.36%, 86.87%, 86.55% and 96.24% respectively, which were higher than 41.19%, 49.90%, 13.46% and 62.65% respectively in 2014, difference were all significant (all P < 0.001). From 2014 to 2020, HH compliance rate (β=6.248, P < 0.001), bed-day consumption of alcohol-based hand rub + hand sanitizer (β=1.182, P=0.013) and paper towel(β=0.508, P < 0.001) increased year by year, case incidence of HAI (β=-0.326, P < 0.001) decreased year by year, HH compliance rate, bed-day consumption of salcohol-based hand rub + hand sanitizer and paper towel were negatively correlated with incidence of HAI (r=-0.977, -0.793, -0.951), difference were all significant (P < 0.05). Conclusion Application of continuous PDCA cycle to implement bundle intervention measures can effectively improve the compliance of HH and reduce the incidence of HAI.