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Foods and water can be contaminated with antibiotics in China, which may affect children's health, but evidence on antibiotic exposure with precocious puberty (PP) is limited. This study explored the association of antibiotic exposure with PP in a school-based setting. A cross-sectional study with multistage stratified cluster random sampling was conducted in Zhongshan City, Guangdong Province and Qufu City, Shandong Province in China from October 11 to December 5, 2019. A first-morning urine sample was collected to detect antibiotic exposure. We detected 33 of 45 types of antibiotics from eight categories in 928 primary school children aged 6–12 years using HPLS-MS/MS. Detection rate of antibiotics was stratified by sex, study site, and BMI. The Tanner stages were assessed by professional pediatricians from local hospitals. PP is defined as the onset of secondary characters before 8-year-old or menarche before 10-year-old for girls and before 9-year-old for boys. Multivariable logistic regression was performed to examine the association between antibiotic exposure and PP after adjusting potential confounders. The overall detection rate of antibiotics was 93.0% in 928 children. We found the detection rate of tetracyclines and fluoroquinolones in children with PP was significantly higher than that of children with normal puberty (41.4% vs 29.9%, 56.8% vs 50.6%, respectively, all p < 0.05). Both fluoroquinolones (odds ratio (OR): 1.835, 95% confidence interval (CI): 1.066–3.158) and tetracyclines (OR: 2.120, 95% CI: 1.175–3.825) were associated with increased OR of PP after adjusting sex, age, BMI, study site, and family income. Specifically, compared to the values less than the limits of detection, low concentration of ofloxacin from fluoroquinolones (OR: 2.056, 95% CI: 1.091–3.875) and high concentration of chlortetracycline (OR: 3.027, 95% CI: 1.126–8.140) and tetracycline from tetracyclines (OR: 2.756, 95% CI: 1.167–6.506) were associated with increased OR of PP. Exposure to antibiotics, especially fluoroquinolones and tetracyclines was positively associated with precocious puberty. • First study on antibiotics exposure in precocious puberty by HPLC-MS/MS. • 928 primary school children aged 6–12 years were included. • 45 types of antibiotics were measured using HPLC-MS/MS. • Antibiotic level was major contributor to overall variance in risk estimate. [ABSTRACT FROM AUTHOR]