Objective To investigate the prevalence of nosocomial infection among inpatients in a tertiary hospital， and provide scientific evidence for hospital infection control and targeted surveillance. Methods A cross-sectional survey was conducted to investigate a total of 543 hospitalized patients using the hospital information system. Results The prevalence of hospital-acquired infection （HAI） was 4.24%， and that of community-acquired infection （CAI） was 23.39%. HAI prevalence differed significantly among the departments （ χ ²=148.870， P <0.05）， and was highest in the department of intensive care medicine （72.73%）. Sites of infection were significantly different between HAI and CAI （ χ ²=22.942， P =0.011）； however， the most frequent site of infection was lower respiratory tract in both HAI and CAI. Major pathogens for nosocomial infection were Gram-negative bacteria （56.92%）， mainly Escherichia coli and Klebsiella pneumoniae . Antimicrobial usage was observed in 31.68% of the patients， principally for therapeutic use of antibacterial drugs （80.23%） and a combination of drugs （88.95%）. Examination rate of pathogens following the antimicrobial usage was 72.08%. Conclusion The investigation on the prevalence of nosocomial infection may facilitate fully understanding the nosocomial infection. It warrants strengthening the monitoring in the departments of intensive care medicine and on multi-resistant bacteria， and achieving antimicrobial stewardship， so as to improve the awareness of hospital infection control in medical staff' and effectively reduce the occurrence of nosocomial infection.