Abstract: Graphene oxide (GO) was prepared by the modified Hummers method and used as a fluorescence quenching reagent of L-tryptophan (L-Trp). The structure of GO was characterized by FT-IR and Raman spectroscopy. The binding mechanism of L-Trp on GO and their reaction were investigated by UV spectroscopy and the fluorescence method. Results show that GO has a strong fluorescence quenching ability for L-Trp. The quenching efficiency is 95.6% for L-Trp with a concentration of 2×10^-5 mol·L^-1 when the GO concentration is 16×10^-5 g·mL^-1. The binding constants of L-Trp on GO were 2.03×10⁵, 1.83×10⁵ and 1.63×10⁵ mL·g^-1 at 25, 30 and 35℃, respectively, as revealed by the Lineweawer-Burk equation, indicating a strong interaction between GO and L-Trp. GO quenches the intrinsic fluorescence of L-Trp by the formation of a complex, and is a static quenching mechanism. The quenching rate constant was 6.65×10¹² mL·(g·s)^-1 at 25℃ as determined by the Stern-Volmer equation.