In recent years, food-derived hypoglycemic peptides have received a lot of attention in the study of active peptides, but their anti-diabetic mechanism of action is not yet clear. In this study, camellia seed cake protein (CSCP) was used to prepare active peptides with α-glucosidase inhibition. The optimization of the preparation of camellia seed cake protein hydrolyzed peptides (CSCPH) was conducted via response surface methodology (RSM) using a protamex with α-glucosidase inhibition as an indicator. The optimal hydrolysis conditions were pH 7.11, 4300 U/g enzyme concentration, 50 °C hydrolysis temperature, and 3.95 h hydrolysis time. Under these conditions, the α-glucosidase inhibition rate of CSCPH was 58.70% (IC₅₀ 8.442 ± 0.33 mg/mL). The peptides with high α-glucosidase inhibitory activity were isolated from CSCPH by ultrafiltration and Sephadex G25. Leu-Leu-Val-Leu-Tyr-Tyr-Glu-Tyr (LLVLYYEY) and Leu-Leu-Leu-Leu-Pro-Ser-Tyr-Ser-Glu-Phe (LLLLPSYSEF) were identified and synthesized for the first time by Liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) analysis and virtual screening with IC₅₀ values of 0.33 and 1.11 mM, respectively. Lineweaver-Burk analysis and molecular docking demonstrated that LLVLYYEY was a non-competitive inhibitor of α-glucosidase, whereas LLLLPSYSEF inhibited α-glucosidase, which displayed a mixed inhibition mechanism. The study suggests the possibility of using peptides from Camellia seed cake as hypoglycaemic compounds for the prevention and treatment of diabetes.