Objective To evaluate the effect of comprehensive intervention measures on hand hygiene (HH) compliance of health care workers (HCWs) with the interrupted time-series (ITS) analysis. Methods Staff from all clinical and technical departments of a tertiary hospital were selected as the research subjects, during the preparation period for the tertiary hospital accreditation, HH comprehensive intervention measures, including training, investigation, competition, and etc.were implemented.The monitoring data of HH compliance before and after intervention were collected, and the ITS analysis was applied to construct a model for evaluating the immediate and long-term effect of intervention. Results HH compliance rate of HCWs increased from 67.58% before intervention to 81.43% after intervention.ITS analysis result showed that overall HH compliance rate of HCWs increased by 19.717%(t=5.473, P < 0.001) immediately after the intervention, but the slope change was-1.995(t=-2.249, P=0.048), indicating an expanding downward trend in HH compliance after implementation of intervention.The intervention had immediate improvement effect on staff with different jobs, with the doctor group having the greatest improvement.The slope change for staff with different jobs was negative, with the absolute value of the change for the nurse group being the smallest. Conclusion Comprehensive intervention measures can effectively improve HH compliance of HCWs, but the long-term effect is not ideal.ITS model can be effectively used to analyze the monitoring data of HH compliance.