Objective: To investigate the knowledge, compliance and the influencing factors of hand hygiene of multiple provincial medical centers’ staffs, and explore the factors affecting compliance, to provide the scientific basis for improvement of hand hygiene. Methods: All categories of medical personnel were observed and recorded by using single-blind method from twelve clinical departments of five top hospitals in Hunan Province to survey their hand hygiene compliance, and inquired with questionnaire. Chinese modified version of De Wandel D’s questionnaire based on a behavioral theory model was filled out by staffs to reveal the influencing factors. Results: The hand hygiene practices of 416 medical personnel（143 doctors,192 nurses, 81 care workers and health cleaning staffs）were observed and recorded. Total rate of hand hygiene compliance (HHC) of medical personnel was 47.78%, compliance of nurses was 58.95%, physicians was 41.96%, care workers and health workers was only 21.62%(P<0.001). The rates of HHC “Before direct contact with the patients”, “before operation or nursing program”, “After blood or body fluids exposure”. “after contacting with patients”, “after contact with the surroundings of patients” were 27.59%, 49.32 %,63.37%, 61.68% and 38.62% respectively. ICU, neonatology and transplant center’s hygiene compliance was significantly higher than other departments.The questionnaire shows the lack of realization of hand hygiene, tight working hours, lack of hand-washing facilities, less of humane care, and poor self-discipline were affecting factors of hand hygiene compliance; Hand hygiene knowledge of auxiliary was urgent need to strengthen. Conclusion: The compliance rate of HH of medical personnel in t Hunan’s top hospitals needs to be improved. The pertinent measures to improve HH should be taken according to enhance hand hygiene knowledge, raise realization, improve handwashing facilities, and strengthening the humane care and management.