Antibiotics are extensively used for health protection and food production, causing antibiotic pollution in the aquatic environment. This study aims to determine the bioavailability and bioaccumulation of typical antibiotics sulfamethoxazole (SMX) and roxithromycin (RTM) in zebrafish under environmentally realistic conditions. Four different microcosms, i.e. water, water-sediment, water-zebrafish, and water-sediment-zebrafish were constructed, with three replicates in parallel. The concentrations of SMX and RTM in water, sediment and zebrafish were extracted and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to assess their kinetic behavior and bioavailability. In the water-sediment system, the dissolved concentration of both SMX and RTM decreased with time following the first-order kinetic while their adsorption by sediment increased with time. In the water-zebrafish system, SMX and RTM bioaccumulation was increasing with time following the pseudo second-order kinetics. RTM bioaccumulation in zebrafish (up to 16.4 ng/g) was an order of magnitude higher than SMX (up to 5.2 ng/g), likely due to RTM being more hydrophobic than SMX. In addition, the bioaccumulation factor (BAF) value of SMX in zebrafish was greater than its sediment partition coefficient, while the opposite trend was observed for RTM, demonstrating the importance of antibiotics properties in affecting their bioavailability. Furthermore, increasing dissolved organic carbon concentration in water reduced SMX bioaccumulation, but increased RTM bioaccumulation at the same time. The findings are important in future studies of environmental fate and bioavailability of toxic chemicals with different pollution sources and physicochemical properties.