ObjectiveTo investigate healthcareassociated infection (HAI) in neonatal intensive care unit(NICU), and provide reference for controlling HAI in newborns. MethodsTargeted surveillance on HAI in all newborns in NICU of a hospital from January 2011 to December 2013 was performed by  prospective investigation method.  ResultsA total of  5 365 newborns were monitored, 128 newborns developed 133 times of HAI,HAI rate and HAI case rate was 2.39% and 2.48% respectively. The major infection sites were conjunctiva (33.08%), upper respiratory tract (23.31%) and  skin soft tissue (17.29%). The daily infection rate was 3.32‰(128/38 528), vascular catheter usage rate and respirator usage rate was 1.75%（676/38 528）and 1.09%（420/38 528）respectively. The difference in vascular catheter usage rate and respirator usage rate in newborns with different body weight had statistical difference (χ2=3 075.22; 442.46, both P＜0.01）. HAI in newborns with body weight of ≤1 000 g, 1 001-1 500 g, 1 501-2 500 g, and ＞2 500 g was 16.67%,12.26%,4.15% and 1.06% respectively. None newborns developed catheterrelated bloodstream infection(CRBSI), the incidence of ventilator-associated pneumonia (VAP) was only 4.76‰（2/420）. A total of 51 specimens were sent for detection, 27 pathogens were isolated, the major were grampositive bacteria（55.56%）.ConclusionThe incidence of HAI in newborns is high,  the main infection sites are eye, upper respiratory tract, and skin and soft tissue.