Background: Misconceptions about antibiotics among the public can potentially lead to inappropriate use of antibiotics. Currently, there is no antibiotic knowledge assessment tool to address this issue. This study aimed to develop and validate an Antibiotic Knowledge Scale (AKS) to assess public knowledge of antibiotics in China. Methods: An initial 18-item AKS was designed and validated among 1,180 people recruited in June 2017. After removing redundant items, the reliability and validity of the AKS were examined. Subsequently, a cross-sectional Internet survey was conducted in China, and 12,772 people were recruited using convenience sampling and surveyed using a structured anonymous questionnaire. A logistic regression model was used to identify the factors associated with poor knowledge of antibiotics. Findings: The final AKS included two screening items and fifteen knowledge evaluation items. Cronbach's alpha, test-retest reliability, and split-half reliability were 0.90, 0.88, and 0.89, respectively. A confirmatory factor analysis indicated that the AKS has good construct validity. The mean score on the AKS was 7.25 (SD = 4.76) and 67% of participants had poor antibiotic knowledge, which was associated with male gender, rural residence, lower educational level, poor economic status, living in Western China, and lacking education on antibiotics. Interpretation: The AKS demonstrated satisfactory reliability and validity. The majority of participants had inadequate knowledge of antibiotics, and the government urgently need conduct interventions to improve public knowledge of antibiotics. Funding: This research was funded by National Natural Science Foundation of China (No.0204513254). Declaration of Interest: The authors declare no competing interests. Ethical Approval: Ethical approval was provided by the Human Research Ethics Committee, Huazhong University of Science and Technology, Wuhan, China.