Objective To investigate the status of nurses’ cognition of skin test for β-lactam antibiotics in tertiary hospitals in China and its influencing factors. Methods The nurses in the eastern and western regions were selected by stratified sampling and convenient sampling between March and May 2023. A multi-center survey was conducted with the help of the questionnaire star platform using the “Questionnaire on the Cognitive Status of β-lactam Antibiotics Skin Test” developed by the “Guidelines for β-lactam Antibiotics Skin Test (2021 Edition)” issued by the National Health Commission of the reference country. Results A total of 89 tertiary hospitals were surveyed. Among them, there were 27 in the eastern regions and 62 in the western regions; There were 64 comprehensive hospitals and 25 specialized hospitals. A total of 4 241 nurses were included. Among them, 4 151 were females and 90 were males. The scores of nurses’ cognition of skin test of β-lactam antibiotics were (61.47±16.27). The survey results showed that the accuracy rate ranges from 15.52% to 88.98%. Only no routine skin test before cephalosporin administration, judgment of skin test results, and infusion device replacement after allergy were greater than 80%. The results of the logistic regression analysis showed that the nurses with professional titles and antimicrobial management training in the past 2 years were independent influencing factors of nurses’ cognition of skin test of β-lactam antibiotics (P<0.001). Conclusions At present, nurses in tertiary hospitals in China still have insufficient knowledge of skin test of β-lactam antibiotics. It is suggested that hospitals should form corresponding hospital skin test of β-lactam antibiotics standards in combination with guidelines and strengthen training for nurses in this regard, so as to improve nurses’ correct cognition and implementation of skin test of β-lactam antibiotics.