Human serum albumin (HSA) and human growth hormone (hGH) fusion protein [HSA/GH] is a promising long-acting form of GH to treat GH deficiency. This study attempted to engineer a P. pastoris strain for high-level production of HSA/GH to be used in basic research and clinical application. Strains contained two, three, and seven copies of HSA/GH gene were screened by selecting against Zeocin resistance. The results revealed that introducing two to three copies of HSA/GH gene was sufficient to give a significant increase in secretion level, compared with one copy of HSA/GH gene. No significant differences were observed between two to three copies and seven copies. Co-expression with either one copy of exogenous ERO1 or PDI in a strain carrying multicopies of HSA/GH gene led to varying degrees of increase in HSA/GH secretion. The effect of introducing multicopies of PDI was similar to that of one copy of PDI, but introducing excess copies of ERO1 reduced HSA/GH secretion. Simultaneous co-expression with PDI and ERO1 was less effect than either PDI co-expression or ERO1 co-expression. A strain showing higher secretion level was successfully applied to large-scale fermentation with the productivity of 3-4 g/l.