The massive use of antibiotics and the consequent antibiotic pollution have become a major risk to river ecosystems, water quality, and human health. In this study, 28 antibiotics were analyzed in the rural-urban river water of Suzhou City, the inflow rivers of Hongze Lake. The total concentration of antibiotics in the rural-urban river was nd-85.87 ng/L, mainly containing veterinary antibiotics, quinolones, and tetracyclines. The distribution of antibiotics was found to be closely related to the population density and the development of the breeding industry along the bank through structural equation model and correlation analysis. Meanwhile, the human activities (70%), livestock and poultry breeding (30%) were the main sources of antibiotics in the study area through the principal component analysis-multiple linear regression (PCA-MLR) source analysis. Finally, the risk assessment was carried out by the Risk Quotient (RQ) method, and indicated that antibiotics pose a low risk to plant and invertebrates ,but Ofloxacin, Ciprofloxacin, Erythromycin, Norfloxacin, and Chlortetracycline posed a mid ecological risk to algae. This work provide a valuable data set across the rural-urban river network and revealed the antibiotic contamination profile. Prevention and management to reduce the antibiotic inputs are needed for the rural-urban zones.