The bioavailability and biomagnification of organophosphate esters (OPEs) were investigated in a food web in the Zhushan Bay of Taihu Lake, China. The organisms included mainly three biological groups: plankton, invertebrates, and fish, which displayed distinctly different compositional profiles of OPEs. In general, the log BAFs (bioaccumulation factor) of OPEs displayed a significant correlation with their log K_ow (octanol-water partitioning coefficient), suggesting that the bioaccumulation was mainly controlled by the hydrophobicity. The log BAFs of the more hydrophobic OPEs in benthic invertebrates were higher than in fish, suggesting that ingesting sediment constituted additional exposure route for benthic invertebrates. The log BSAFs (biota-sediment accumulation factor) in the benthic invertebrates increased with log K_ow in the range of 1.44–5.73 and decreased afterwards. The depressed bioavailability of the highly hydrophobic OPEs was attributed to their strong adsorption to the sediment. The biomagnification potency of OPEs was affected by hydrophobicity of the compounds and biotransformation properties in the organisms at different trophic levels. 2-Ethylhexyl diphenyl phosphate biomagnified in the fish food web of Taihu Lake with a TMF (trophic magnification factor) of 3.61, which was due to the combined results of its relatively high hydrophobicity (log K_ow of 5.73) and decreased metabolism potential in the high-trophic-level fish. The constant metabolism diminished the biomagnification potency of hydrophobic compounds triphenyl phosphate and tricresyl phosphate in this food web.