Abstract: α-Amylase inhibitors play a pivotal role in modulating postprandial glycemia, and food-derived bioactive peptides are attracting increasing attention due to their favourable safety profiles. This study aimed to screen for an efficient α-amylase inhibitory peptide from white kidney bean (Phaseolus vulgaris L.; Pv-αAIP), and to elucidate its structure–activity relationship as well as inhibitory mechanism. Pv-αAIP was prepared by acidic protease hydrolysis, followed by fractionation using gel permeation chromatography and ultrafiltration to isolate high-activity fractions. The most active fractions were screened, and target candidate peptides were identified by LC–MS/MS. Molecular docking was employed to probe the inhibitory mechanism and their gastrointestinal stability was predicted based on sequence characteristics. Results showed that the 1~10 kDa fraction (Pv-αAIP-4) exhibited the strongest inhibitory activity (79.31%). Structural characterisation of Pv-αAIP-4 revealed the following: UV–Vis spectroscopy exhibited stronger absorption near 280 nm; FTIR spectra displayed more regular band shapes with fewer extraneous peaks near 2930 cm⁻¹, and within the 1620~1640 cm⁻¹ as well as 1400~1450 cm⁻¹ regions; circular dichroism analysis revealed secondary structure contents of β-sheet, α-helix, and random coil at 33.69%, 40.67% and 5.50%, respectively; scanning electron microscopy revealed a uniform, porous network morphology. Thirteen candidate peptides were obtained by LC–MS/MS and subsequent feature selection. Molecular docking against human α-amylase (PDB ID: 3BAJ) demonstrated that all candidates had binding energies superior to acarbose (−6.9 kcal/mol). Notably, the long peptide DPWGGFPFPSPHTP (−9.7 kcal/mol; 13 hydrogen bonds) and the short peptide FNLF (−7.9 kcal/mol; 6 hydrogen bonds) engaged the catalytic centre through multipoint interactions and a precise anchoring mechanism, demonstrating pronounced inhibitory potential. Sequence-based gastrointestinal stability analysis further suggested that both peptides have favorable prospects for oral applicability, with the proline-rich long peptide exhibiting higher predicted stability. These findings provide a theoretical basis and candidate molecules for the development of natural hypoglycemic peptides and the creation of functional foods.