Objective To estimate the incidence of healthcare-associated infection(HAI) based on the survey results of prevalence rate of HAI in a hospital, and provide scientific basis for key interventions to reduce the incidence of HAI. Methods The survey data of HAI prevalence rate in a tertiary first-class hospital in 2018 were retrospectively analyzed, Rhame and Suddertth formula was used to estimate the incidence of HAI in different departments and at different sites. Results Prevalence rate and estimated incidence of HAI were 3.84% and 4.57% respectively. Prevalence rate of HAI in intensive care unit was the highest (16.13%), the estimated incidence in department of pediatrics was the highest (8.78%). Prevalence rate and incidence of HAI in lower respiratory tract was the highest (2.35% and 2.28% respectively). Conclusion It is suggested that survey on prevalence and estimated incidence of HAI should be reported at the same time, so as to improve the scientific nature of intervention measures and reduce the occurrence of HAI.