ObjectiveTo investigate the prevalence of healthcareassociated  infection(HAI)  in a children’s specialty hospital in Guangzhou in 2014.MethodsA crosssectional survey was undertaken to investigate the prevalence of HAI among all hospitalized patients on September 17, 2014.ResultsA total of 997 patients were investigated, 30 patients developed 32 times of  HAI, HAI rate and HAI case rate were 3.01% and 3.21% respectively. The main infection sites were upper respiratory tract（n=11，34.38%）；the departments with higher HAI prevalence rates were intensive care unit(ICU, 3.99%) and  pediatric internal medicine department（3.60%）; the rate of etiological examination in patients with HAI accounted for 93.33%, a total of 24 strains of pathogens were isolated, 6 of which were virus (25.00%), 6 were fungi (25.00%), 11 were bacteria (45.83%), and 1 was Chlamydia (4.17%). The main bacteria were Staphylococcus aureus (n=3, 27.27%) and Staphylococcus epidermidis (n=3, 27.27%); a total of 451 (45.24%) patients received antimicrobial agents on the investigation day,  67.41% of whom received therapeutic use of antimicrobial agents，19.73% received prophylactic use, and 12.86% received both therapeutic and prophylactic use; monodrug application accounted for 82.26%, the percentage of bacterial detection among patients with therapeutic antimicrobial use (including  combination of therapeutic and prophylactic use) was 82.60%.ConclusionManagement of key departments and key sites of HAI should be strengthened in children’s specialty hospital, antimicrobial agents should be used rationally, so as to protect the safety of children.