Background: This study aimed to investigate the prevalence of healthcare-associated infection (HAI) in the China-Japan Friendship Hospital, a tertiary level hospital in Beijing, China. Methods: We defined HAI using the criteria established by the Ministry of Health of the People's Republic of China. Three cross-sectional surveys were conducted from 2012 to 2014. Inpatients who had been hospitalized for at least 48 h were surveyed. Information on HAI prevalence, isolated pathogens and use of antibiotics were collected. Logistic regression models were used to assess the associations between HAI and potential risk factors. Results: During three cross-sectional surveys, a total number of 4,029 patients were included (1,233 patients in 2012, 1,220 patients in 2013 and 1,576 patients in 2014). The overall prevalence of patients with HAI was 3.6 % (95 % confidence interval (CI) 3.1 %-4.2 %). Respiratory tract infections were the most common type (64.7 %) of HAIs, followed by urinary tract infections (12.6 %) and bloodstream infections (5.4 %). HAI occurrences were significantly associated with male sex (odds ratio (OR) = 2.25, 95 % CI 1.53-3.32), age over 85 years (OR = 4.74, 95 % CI 2.54-8.83), hospitalization in the intensive care units (ICUs) (OR = 2.42, 95 % CI 1.31-4.49), indwelling urinary catheter (OR = 4.21, 95 % CI 2.46-7.20) and mechanical ventilation (OR = 2.31, 95 % CI 1.30-4.09). Gram-negative bacteria were found to be the most isolated pathogens (67.1 %), with gram-positive bacteria and fungi accounted for 20.3 % and 10.5 %, respectively. Antibiotics were administered to 34.3 % of the included patients over the study period. Conclusions: The overall HAI prevalence in our hospital is similar to previous studies that were conducted in other areas of China, and the respiratory tract infection should be the priority in HAI reduction control within China. We should focus HAI reduction efforts on patients with advanced age, hospitalization in the ICU and indwelling devices.