Interactions of tetracycline (TC) with dissolved organic matter (DOM) collected from Lake Taihu and Lake Xuanwu were investigated through fluorescence quenching. Three fluorescent DOM components, fulvic-(C1), tryptophan-(C2), and tyrosine-like (C3) component, were identified in DOM via excitation emission matrix spectra with parallel factor analysis (EEM-PARAFAC). Results from fluorescence quenching titration showed that the fluorescence of 3 components was quenched at different degree by TC through static quenching. When the TC concentration increased to 45.5 μmol/L, the whole florescence of C2 and C3 were almost quenched, while 20% of C1 fluorescence was inaccessible. The quenching also induced conformational changes in DOM molecules. Synchronous fluorescence spectra combined with two dimensional correlation spectroscopy (2D-COS) further revealed that the complexation firstly occurred for tryptophan-like substance, followed by tyrosine-and fulvic-like substance. The associated complexing constants (lgK) of 3 components with TC were in the range of 5.05-5.85, followed the order of C2 > C3 > C1. The strong binding ability of protein-like substances confirmed their prominent role in the TC-DOM complexation. These results highlight the significant impact of DOM on the bioavailability and ecotoxicity of antibiotics in freshwater lakes.