Affected by point and non-point source pollution, the Fenhe River Basin faces significant environmental challenges. This study aimed to analyze the distribution characteristics and influencing factors of antibiotics in the water and sediments of the Fenhe River Basin. Samples were collected from 23 sites within the basin, and 26 antibiotics from five different classes were detected and analyzed using high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS). The water–sediment partition coefficient (Kp) was calculated, and spatial analysis was conducted using geographic information system (GIS) technology. The results showed that 25 antibiotics were detected in the water, with concentrations ranging from 130 to 1615 ng/L, and 17 antibiotics were detected in the sediments, with concentrations ranging from 121 to 426 μg/kg. For quinolones (QNs), except for ofloxacin, all others could be calculated with overall high values of Kp ranging from 692 to 16,106 L/kg. The Kp values for QNs were generally higher in the midstream, with considerable point source pollution from industries and non-point source pollution from developed agriculture. The distribution of Kp is closely associated with risk. This study found that the Kp values of the antibiotics were influenced by various factors such as temperature, water flow, and the physicochemical properties of sediments. Correlation analysis revealed significant relationships between Kp and parameters such as river width, water depth, water quality (total nitrogen, total phosphorus, and chemical oxygen demand), and sediment pH and clay content.