The interaction between riboflavin and egg white riboflavin binding protein (RBP) was investigated using fluorescence spectroscopy. The binding mode, binding constants, thermodynamic parameters between riboflavin and RBP and energy transfer were studied. The experimental results showed that riboflavin has the ability to quench the intrinsic fluorescence of RBP because of a complex formed, and the quenching mechanism is static quenching. The binding constants were 5.35×108, 1.54×108, 0.56×108 L·mol-1 at 298, 308 and 318 K, respectively. The thermodynamic parameters were calculated, which suggested hydrogen bonds and Van der Waals played a major role in the interaction. The distance and efficiency of energy transfer between riboflavin and RBP were 0.70 nm and 0.39, respectively, based on the theory of Frster nonradiative energy transfer. Furthermore, the synchronous fluorescence spectroscopy was utilized to investigate the conformational transformation.