Graphene oxide was prepared by the modified Hummers method and characterized by field emission scanning electron microscopy. The interaction of graphene with methylene blue was studied by UV absorption, the intensity of two main absorption peaks of methylene blue decreased significantly after the fluorescence was quenched, and the energy transfer didn’t occur because the overlap of the absorption spectrum of GO and the emission spectrum of MB is too small. Therefore, the fluorescence quenching of MB and GO was static. When adding a certain amount of Bi3+ in the graphene-methylene blue system, Bi3+ replaces the methylene blue from the graphene-methylene blue complexes because Bi3+ has the smaller volume and is more positively charged. The methylene blue therefore dissociates from the GO-MB complexes, resulting in the recovery of fluorescence of the system. Furthermore, the fluorescence of the system increases with the increase in the amount of Bi3+ due to the enhanced amount of MB in the system. A novel spectrofluorimetric method was therefore developed for the sensitive determination of Bi3+. Some parameters including the concentration of methylene blue, the amount of graphene oxide, the amount of nitric acid and the sequence of reagent adding were optimized to obtain higher sensitivity. The fluorescence of the system was detected at an emission wavelength of 667 nm with excitation at 690 nm. Under the optimized conditions, the concentration of Bi3+ showed good linear relationships with the fluorescence intensity in the range of 0.5～100 μmol·L-1, with correlation coefficients of r=0.995 5. The limits of detection for Bi3+ was 1.0×10－8 mol·L-1 (S/N=3). The selectivity of the proposed method was evaluated and the results showed that 1 000-fold K+, Ca2+, Na+, Mg2+, Cu2+; 100-fold Fe3+, Be2+, SiO2-, Al3+, Ni2+, Sb3+, NO－3, Cl－, F－, and 20-fold Pb2+, Hg2+, Cd2+ had negligible interference with the determination of Bi3+. The method has advantages of sensitivity, rapidness and low cost. It was used for the determination of Bi3+ in environmental water samples including pond water, rain water and sewage water with recoveries of 93.4%～105.2%.