Based on the rDNA sequence of Pichia pastoris, a multi-copy gene expression vector of transglutaminase (pPICZα-rDNA-mtg) was constructed and transformed to the host strain (pGAP9-pro/GS115) expressing pro peptide, to obtain the co-expression strain pro/rDNA-mtg (GS115). Real-time fluorescence quantitative PCR (qPCR) was used to analyze transglutaminase gene copy number in the 4 positive strains. We further studied the effect of gene copy on the enzyme production of recombinant Pichia pastoris as well as high-density fermentation of higher expression strain in a 3-L fermenter. The mtg copy numbers of the 4 positive strains were 2.21, 3.36, 5.72 and 7.62 (mtg-2c, mtg-3c, mtg-6c and mtg-8c), respectively, and the enzyme production capacity and protein expression level were mtg-3c>mtg-2c>mtg-6c>mtg-8c. Mtg-3c and mtg-6c of high-density fermentation had the highest enzymatic activity and enzymatic activity per unit wet weight in the supernatant of 3.12 U/mL, 52.1 U/g (wet weight) and 2.07 U/mL and 36.5 U/g (wet weight), respectively. In terms of enzyme activity per unit wet weight, mtg-3c is 1.4 times higher than that of mtg-6c. The activity of purified enzyme (mtg-3c) was up to 7.21 U/mL and the protein concentration was 437.2 μg/mL. By analyzing the effect of mtg copy number on the enzyme production of recombinant strains, mtg-3c is suitable for the co-expression of two genes (pro and mtg) in pro/rDNA-mtg, and its enzyme activity is related to higher protein secretion of the strain.