Knowledge on runoff transport of manure-sourced antibiotics from farmland soil to aquatic environment is limited due to complexity of hydrological regime and pathways. This study monitored natural rainfalls in sloping orchard plots with free-range chickens, with an attempt to investigate the migration characteristics of typical antibiotics via surface runoff as well as the impact of manure presence. Results showed that rainstorms continuously carried away antibiotics in surface runoff and all target antibiotics including those with high af-finities to soil were detected at the beginning of runoff production. Concentration of antibiotics was found to respond strongly to the instantaneous rainfall intensity, showing consistent fluctuations during rainfalls. Con-centrations of sulfonamides and florfenicol were two orders of magnitude higher than that of tetracyclines and fluoroquinolones. Compared to the control without raising chickens, antibiotics migration was considerably increased with the increased runoff production due to soil surface changes caused by chicken activities. Addi-tionally, dynamics of antibiotic concentration significantly correlated with variations of fluorescent DOM components. Chicken manure-derived DOM mainly contained tryptophan moiety, and laboratory fluorescence quenching test with 2D-COS analysis indicated that all antibiotics interacted more strongly and preferentially with tryptophan than humic-like species. Antibiotics bonded to manure DOM with an affinity corresponding to the significance level of their correlations. In this light, potential use of fluorescence indices based on the established correlations may provide a convenient tool for tracing runoff migration of antibiotics during rainfalls.