ObjectiveTo assess the knowledge, attitude and practice among health care workers(HCWs) in key departments on prevention and control of healthcareassociated infection(HAI), take intervention measures, and provide scientific basis for the prevention and control of HAI in medical institutions.MethodsAll HCWs in departments of pediatrics, respiratory diseases，and emergency were surveyed, change in knowledge, attitude and practice among HCWs with different professional titles on prevention and control of HAI before and after the intervention were compared.Results317 HCWs participated in the survey, there were 275 (86.75%) available questionnaires before intervention and 311(98.11%) after intervention. After intervention, scores for HCWs with senior, intermediate, junior and below professional titles on knowledge were (9.15±2.36）, （7.69±2.05）, and （7.73±2.32）respectively，which were all higher than（6.46±2.12）,（5.30±1.55）,（6.16±1.80） before intervention，difference was statistically significant（all P＜0.05）. After intervention, scores for HCWs of junior and below professional titles on attitude and practice were （91.50±9.26） and （86.40±14.52）respectively，which were both higher than （85.95±13.36） and （76.01±15.25）before intervention，differences were both statistically significant（both P＜0.05）; while scores for HCWs of senior and intermediate professional titles on attitude and practice were not statistically significant（both P>0.05）.ConclusionCompared with the knowledge about HAI prevention and control, more effect needs to be put for achieving change in HCWs’ attitude and practice towards HAI prevention and control; change in attitude and practice among HCWs of junior and below professional titles on HAI prevention and control is easier to be implemented.