Objective To investigate the prevalence of healthcare-associated infection(HAI) of inpatients at all levels of hospitals in Hainan Province from 2014 to 2018, provide scientific guidance for reducing the incidence of HAI in Hainan. Methods Cross-sectional survey method was used to conduct bedside survey and medical record survey for inpatients participated in HAI prevalence survey in Hainan at survey day in 2014, 2016 and 2018, survey data was analyzed with SPSS 20.0 software. Results A total of 17 497, 18 509 and 22 705 patients were surveyed in 2014, 2016 and 2018, HAI prevalence rates were 1.88%-3.26%, showing a downward trend(P < 0.001); there were 1 612 infection sites, the main infection were lower respiratory tract infection (813 times, 50.43%) and urinary tract infection (242 times, 15.01%); general intensive care unit was the department with the highest prevalence rate(22.46%-34.53%). A total of 892 pathogenic strains were isolated, Gram-negative bacteria were the main pathogen(n=664, 74.44%), mainly Pseudomonas aeruginosa(n=169, 18.95%) and Escherichia coli(n=122, 13.68%), Staphylococcus aureus accounted for the highest proportion(n=48, 5.38%) among Gram-positive bacteria. Utilization rates of antimicrobial agents were 32.87%-34.95% in three years, showing a downward trend(P=0.001). Conclusion Although the prevalence rate of HAI in Hainan Province has decreased, it is still necessary to strengthen the prevention and control of key departments, key site, key links and high-risk population, gradually reduce the incidence of HAI.