Objective To understanding the current situation and influencing factors of healthcare-associated infection(HAI)in premature infants, provide theoretical and practical basis for the prevention and control of HAI in premature infants. Methods Premature infants who were admitted to the neonatal ward of a hospital from January 2016 to December 2021 were investigated and analyzed retrospectively. Medical records of premature infants were consulted to collect basic information as well as diagnosis and treatment information of premature infants during hospitalization. Results A total of 3 559 premature infants were included in analysis, including 1 964 males and 1 595 females. The average birth weight was (2 108.66 ± 631.17) g. 109 (3.06%) infants had HAI infection. The main infection types were lower respiratory tract infection (44.04%), bloodstream infection (28.44%), and gastrointestinal tract infection (14.68%). 38 strains of HAI-related pathogens were detected, including 33 strains (86.84%) of Gram-negative bacteria, 3 strains of Gram-positive bacteria, and 2 strains of fungi. The main isolated pathogens were Enterobacter aerogenes (28.95%) and Klebsiella pneumoniae (28.95%). Univariate and unconditional multivariate logistic regression analysis showed that amniotic fluid contamination, birth weight < 2 500 g, ventilator use, and central venous catheterization were independent risk factors for HAI in premature infants (OR values were 2.424, 3.805, 3.776, 5.412, respectively, all P < 0.05). Cesarean section was a protective factor for HAI in premature infants (OR=0.362, P < 0.05). Conclusion Premature infants have a high risk of HAI and multiple influencing factors. Clinical attention should be paid, evidence-based prevention and control measures should be actively adopted to reduce exposure to risk factors and protect the health of premature infants.