Objective To understand the basic situation and characteristics of healthcare-associated infection(HAI) in secondary and higher medical institutions in Guizhou Province, and provide basis for the prevention and control of HAI in all levels of hospitals. Methods By filling out questionnaires, the relevant indexes of HAI in participating hospitals from January 1 to December 31, 2017 were collected and analyzed. Results HAI surveillance data of 234 medical institutions in the whole province were collected, incidence and prevalence rate of HAI were 0.65% and 1.68% respectively, surgical site infection rate and antimicrobial prophylaxis rate in class I incision operation were 0.41% and 37.27% respectively, incidence of catheter-related bloodstream infection(CRBSI), ventilator-associated pneumonia(VAP), and catheter-associated urinary tract infection (CAUTI) per 1 000 days were 1.04‰, 6.81‰, and 1.99‰ respectively. Differences in the indexes of different regions and different levels of medical institutions were all significant (all P<0.05). Incidences of CRBSI, VAP and CAUTI per 1 000 days in patients in tertiary medical institutions were all lower than those of secondary medical institutions(0.92‰ vs 1.23‰, 5.91‰ vs 9.16‰, 1.64‰ vs 2.44‰ respectively, all P<0.001). Conclusion This survey has grasped the characteristics and differences of HAI in different regions and different levels of medical institutions in Guizhou Province, which provides a scientific basis for guiding all levels of hospitals to do well in the prevention and control of HAI. Secondary medical institutions should strengthen the prevention and control of HAI.