Objective To conduct a large-scale survey on the competence of healthcare workers (HCWs) in stan-dard prevention, assess the current status and training needs, and provide basis for the effective implementation of standard prevention. Methods A convenience sampling method was used to conduct an online questionnaire survey among HCWs from various medical institutions across the city. The survey contents included general information, competence questionnaires (basic cognitive and practical competence), as well as training willingness and needs. Results A total of 6 773 HCWs from 62 medical institutions participated in the survey. The average score of their competence in standard prevention was (14.98±2.63) points, with a total scoring rate of 68.07%. The scoring rate of "practical competence" (63.79%) was lower than that of "basic cognition" (73.20%). The highest scoring rate for each dimension was "hand hygiene" (90.90%), and the lowest was "use of protective equipment" (17.25%). Males, technicians, primary medical institutions, college degrees, work experience under 10 years, and emergency/general departments were characteristics of HCWs with lower standard prevention competence. 99.37% of the HCWs were willing to participate in training."once a month" "Monday to Friday afternoon" "15-30 minutes" "online learning" "use protective equipment" "local infection control experts" were the top choices in terms of training needs. Conclusion HCWs’ competence in standard prevention needs to be improved urgently, especially in the aspects of weak practical skills and differences among various groups, HCWs have a strong willingness to participate in standard prevention training. Future efforts should focus on overlooked population in HAI management, increase their training opportunities and diversity, and enhance practice implementation.