Abstract: Objective: To study the effect of galactomannan on the spectral characteristics of human serum albumin (HSA) and the mechanism of their interaction. Methods: Multispectroscopic analyses were used to determine the quenching method, number of binding sites, type of binding force, and changes in secondary structure between galactomannan and HSA. The type and length of binding force were obtained by molecular docking simulation, which further proved that the mechanism of interaction between galactomannan and HSA. Results: Under the action of galactomannan, the endogenous fluorescence of HSA was regularly quenched. The quenching process was spontaneous. The mechanism was static quenching. The number of binding sites was about 1, and the α-helix of HSA was reduced by 7.7%. The results of molecular docking showed that galactomannan interacted in HSA subdomain IIB through hydrogen bonds and van der Waals forces. Conclusions: The interaction between galactomannan and HSA was strong, and the binding was spontaneous.