α-Glucosidase enzymes play a crucial role in digesting starch into glucose, making it a prominent target for diabetes management and the formulation of low-glycemic-index (GI) products. The objective of this research was to develop a Pichia pastoris expression system for the production of three recombinant human α-glucosidases, MGAM-C, MGAM-N, and SI-N, with subsequent screening of high-expression strains, protein purification, and enzymatic analysis. The coding sequences for human MGAM-C, MGAM-N, and SI-N were codon-optimized according to the codon preference of P. pastoris. The constructed plasmids, pPIC9k-MGAM-C, pPIC9k-MGAM-N, and pPIC9k-SI-N, were linearized and transformed into the G115 by electrotransform. After screening using MD and G418, the yeast strains were cultured and induced with methanol. Expression of the target proteins was confirmed by SDS-PAGE and Western blotting. On 10% SDS-PAGE, each of the expressed proteins appeared as a distinct band of approximately 100 kDa, indicating successful translation of the MGAM and SI genes and production of the corresponding recombinant proteins in P. pastoris. Optimization of fermentation conditions revealed that the maximum expression levels were achieved at 72 h, 120 h, and 168 h for GS115-MGAM-C, GS115-MGAM-N, and GS115-SI-N, respectively. The enzymatic activities of MGAM-C, MGAM-N, and SI-N were subsequently measured at 36.56 U/L, 21.15 U/L, and 7.51 U/L following purification. This work provides a reliable P. pastoris expression system for human α-glucosidases, with MGAM-C, MGAM-N, and SI-N successfully expressed and purified, thereby supporting applications in functional foods, glycemic management, and food technology.