Introduction: In China, phlebotomy practice is mostly executed by nurses instead of phlebotomists. Our hypothesis was that these nurses may lack of knowledge on phlebotomy, especially factors influencing quality of blood samples. This study aims to assess the overall nurses' knowledge on phlebotomy to provide reference for improving blood sampling practice in China., Materials and Methods: A survey was conducted involving nurses from 4 regions and 13 hospitals in China. A phlebotomy knowledge questionnaire was designed based on the Clinical and Laboratory Standards Institute H3-A6 guidelines, combining with the situations in China. Descriptive analysis and binary logistic regression analysis were used to analyze the knowledge level and its influencing factors., Results: A total of 3400 questionnaires were distributed and 3077 valid questionnaires were returned, with an effective return rate of 90.5%. The correct rates of patient identification, hand sanitization, patient assessment, tube mixing time, needle disposing location and tube labelling were greater than 90%. However, the correct rates of order of draw (15.5%), definition of an inversion (22.5%), time to release tourniquet (18.5%) and time to change tube (28.5%) were relatively low. Binary logistic regression analysis showed that the correct rates of the aforementioned four questions were mainly related to the regional distribution of the hospitals (P < 0.001)., Conclusions: The knowledge level on phlebotomy among Chinese nurses was found unsatisfactory in some areas. An education program on phlebotomy should be developed for Chinese nurses to improve the consistency among different regions and to enhance nurse's knowledge level on phlebotomy., Competing Interests: Potential conflict of interest: None declared.