Objective To understand the actual situation and changing trend of healthcare-associated infection(HAI), and analyze the risk factors for HAI. Methods A cross-sectional survey was carried out in a tertiary first-class hospital each year from 2014 to 2018, the survey period was one day, all patients in hospital and discharged on that day were investigated, data of HAI in five years were statistically analyzed. Results A total of 9 718 patients were investigated, prevalence rates of HAI and HAI cases were 4.55% and 5.01% respectively, prevalence rates of HAI and HAI cases decreased year by year in five years, differences were all statistically significant (all P<0.001). Lower respiratory tract was the main infection site(n=193, 39.63%), followed by surgical site (n=75, 15.40%), urinary tract (n=65, 13.35%) and upper respiratory tract (n=29, 5.95%). Department of hematology had the highest prevalence rate of HAI (15.73%), intensive care unit had the highest prevalence rate of HAI cases (16.72%). A total of 414 pathogens were isolated from patients with HAI, including 258 strains of gram-negative bacteria, 112 gram-positive bacteria, 38 fungi and 6 other pathogens. The most common pathogens were Pseudomonas aeruginosa (51 strains), followed by Escherichia coli (49 strains), Acinetobacter baumannii (43 strains), Staphylococcus aureus (43 strains) and Klebsiella pneumoniae (42 strains). Conclusion Investigation on prevalence of HAI is conducive to understanding the incidence of HAI, prevention and control of HAI are the key points of device-related infection and surgical site infection.