Objective To investigate the characteristics of healthcare-associated infection(HAI) in neonatal intensive care unit (NICU), and provide scientific basis for the prevention and control of HAI in NICU. Methods The occurrence of HAI in newborns who were hospitalized in NICU for >48 hours from January 2013 to December 2017 was monitored, and characteristics of HAI and types of pathogens were analyzed. Results From January 2013 to December 2017, a total of 10 551 newborns in NICU were monitored, 257 newborns had 270 cases of HAI; incidence and case incidence of HAI were 2.44% and 2.56% respectively, incidence of HAI per 1 000 day was 2.31, There was no significant difference in the incidence of HAI between different genders (P>0.05). Incidence of HAI in newborns with different birth weight as well as with or without tracheal intubation was significantly different (all P<0.001). Case incidences of HAI among different years were significantly different (P<0.05). The main HAI site was lower respiratory tract, accounting for 65.19% (ventilalor-associated pneumonia accounted for 36.30%), followed by blood stream(22.22%). A total of 169 strains of pathogens were isolated, mainly gram-negative bacteria, the top three were Acinetobacter baumannii (26.03%), Klebsiella pneumoniae (22.49%) and Escherichia coli (19.53%). Conclusion Low birth weight and invasive operation are risk factors for HAI in newborns in NICU. Reducing invasive operation, strictly mastering various indications of catheterization, strengthening prenatal education and nutritional guidance, and maintaining fetal birth weight in a reasonable range are the key to prevent HAI in NICU patients.