Abstract: α-Galactosidase can specifically hydrolyse α-1,6-galactosidic bonds in galactooligosaccharides, polysaccharides, and glycoproteins. This property enables its extensive application in the food industry, animal feed, and biomedicine sectors. In order to achieve efficient heterologous secretion and expression of α-galactosidase from Aspergillus niger in Pichia pastoris, the signal-peptide-deleted α-galactosidase gene aglC was successfully cloned and expressed using Aspergillus niger cDNA as the template. The expression level of aglC in Pichia pastoris was enhanced through gene dosage optimization and the addition of transcription factors associated with protein expression, followed by the determination of its enzymatic properties. Results showed that this α-galactosidase could be secreted in Pichia pastoris, with an initial enzyme activity of 6.7 U/mL. The recombinant enzyme exhibited optimal activity at pH 6.0 and 50°C. When the copy number of the aglC gene was increased to 6, the enzyme activity was elevated to 26.0 U/mL, which represented a 3.9-fold increase compared with that of single-copy fermentation. Screening of transcription factors related to protein expression revealed that co-expression of HAC1 significantly boosted the α-galactosidase yield, with 41.3 U/mL achieved in shake flask fermentation. For the 6C-HAC1 strain, an enzyme activity of 5083.8 U/mL was detected in the supernatant after 240 hours of high-density fermentation. This study established a multi-copy expression strain of α-galactosidase in Pichia pastoris, which effectively enhanced the yield of Aspergillus niger α-galactosidase. This achievement lays a foundation for the application of the enzyme in food processing, animal feed, and biopharmaceutical industries.