Abstract: To investigate the inhibitory effect and mechanism of sea buckthorn extract (SBE) on α-amylase, and to evaluate the influence of acarbose and casein on its inhibitory activity, SBE was prepared using ultrasound-assisted extraction. The inhibitory activity and underlying mechanism were systematically studied through enzyme inhibition assays, enzyme kinetics analysis, fluorescence quenching spectroscopy, and molecular docking, and were complemented by combined inhibition assays and casein-SBE interaction analyses to assess its performance when used with acarbose and under the presence of casein. The results showed that SBE exhibited significant inhibitory activity against α-amylase, with an IC₅₀ value of 88.9±1.2 μg/mL. The inhibition type was identified as mixed-type, involving both competitive and non-competitive modes, and the inhibitory effect of SBE was superior to that of acarbose. Fluorescence quenching analysis revealed that SBE spontaneously formed a complex with α-amylase, altering the microenvironment of the enzyme's chromophores and leading to intrinsic fluorescence quenching, thereby reducing enzymatic activity. Molecular docking further confirmed that the active compounds in SBE could form stable interactions with amino acid residues of α-amylase. Combined inhibition experiments indicated an additive effect between SBE and acarbose. Moreover, casein was found to reversibly interfere with the inhibitory activity of SBE. These findings provide a theoretical basis for the potential development of SBE as a natural α-amylase inhibitor.