Objective To understand the prevalence rate and changing trend of healthcare-associated infection(HAI) in a hospital from 2016 to 2018, and evaluate the effect of HAI management and control measures, so as to provide reference for improving HAI management level. Methods A cross-sectional survey was conducted through combination of bed-side investigation and medical record reviewing, prevalence of HAI among all inpatients in a hospital on November 24, 2016, April 27, 2017, and May 23, 2018 were investigated. Results A total of 790 patients were surveyed during three years, 12 patients had 13 cases of HAI, HAI rate and HAI case rate were 1.52% and 1.65% respectively;HAI prevalence rates of each year were 1.84%, 1.43%, and 1.36% respectively, difference was not significant (χ²=0.208,P=0.935); the main HAI site was lower respiratory tract (46.16%). The utilization rate of antimicrobial agents in the survey day was 53.67%, utilization rate of antimicrobial agents decreased from 69.12% in 2016 to 37.07% in 2018, difference was significant (χ²=57.554,P<0.001). Therapeutic use of antimicrobial agents accounted for 70.91%-79.33%, antimicrobial single use accounted for 73.33%-80.73%, bacterial culture rate for therapeutic use of antimicrobial agents increased from 38.85% in 2017 to 59.30% in 2018, there were significant differences in antimicrobial use purpose, combined antimicrobial use and bacterial culture for therapeutic antimicrobial use in each year (χ²=62.266, 69.774, 8.930, respectively, all P<0.05). Conclusion The prevalence of HAI has not changed significantly in the past three years, antimicrobial use has been standardized year by year, but bacterial culture rate for therapeutic antimicrobial use still needs to be improved.