Objective To analyze the influencing factors for the knowledge level, cognition willingness and attitude of health care workers (HCWs) towards monkeypox. Methods A cross-sectional survey was conducted, from January to April 2023, 700 HCWs were randomly selected from a tertiary hospital in Shanghai. Based on a self-designed questionnaire, the survey was conducted anonymously through the online platform "Wenjuanxing". Results A total of 612 questionnaires were collected, 581 were valid, with a response rate of 87.43% and an effective rate of 94.93%. The mean score of the questionnaire was (128.80±27.70) points, with a score rate of 58.54%. Multiple linear regression analysis showed that there were statistically significant differences in the knowledge level of monkeypox among HCWs of different ages, occupations, departments, educational levels, years of working experience, professional titles, initiative cognition on monkeypox, and participation in monkeypox-related lectures organized by the units (all P < 0.05). Conclusion HCWs' cognition on monkeypox is at a moderate level. The training of HCWs on monkeypox knowledge should be strengthened to improve the emergency response capabilities during monkeypox outbreaks.