Alpha arbutin is a botanical extract which can relieve cough and asthma, and its effects on protein and the mechanism of action are poorly reported. This paper studied the interaction between alpha arbutin and human serum albumin (HSA) under different temperature conditions with fluorescence spectroscopy. With the increasing of alpha arbutin concentration, the fluorescence intensity of HSA was significantly enhanced with the occurrence of blue shift of fluorescence spectrum. By using fluorescence enhancement effect equations, we can obtain the binding constants of alpha arbutin and HSA. Thermodynamic parameters were obtained using the van’t Hof equation, resulting in the main type of molecular force between alpha arbutin and HSA. The thermodynamic parameters(ΔH=-23.29 kJ·mol-1 and ΔS=40.96 J·mol-1·K-1) indicated that hydrogen bond and hydrophobic were the intermolecular force. UV-Vis, Synchronous fluorescence spectrometry and Three-dimensional fluorescence spectra were utilized to explore the effects of arbutin and HSA interactions on the secondary structure of protein. The circular two color spectrometry can conclude the protein secondary structure, which indicated the loss of helical stability after the interaction of arbutin and HSA. The binding details between arbutin and HSA were confirmed by molecular docking, which revealed alpha arbutin was bound at siteⅡ (subdomain IIIA) via multiple interactions, such as hydrogen bond, hydrophobic. The full basic data in the work is to clarify the binding mechanism of alpha arbutin with HSA and is useful for understanding its effect on protein function during the blood transportation process.