Porcine follicle-stimulating hormone (pFSH), comprising and subunits, is commonly used to induce superovulation in domestic animals in assisted reproduction technologies; however, the practical application of pFSH is inhibited by the limited efficiency of its production. Recombinant yeast-derived FSH offers a practical alternative; however, the heterologous expression efficiency remains disappointingly low. To improve FSH production in Pichia pastoris, a series of molecular strategies, together with fermentation optimization, were tested in the present study. By comparing clones of the Mut(s) phenotype strain, it was observed that the yield of soluble pFSH increased by approximately 96% in clones of the Mut(+) phenotype strain. The protein levels of soluble pFSH, which confers biological specificity, increased by approximately 143 and 22% after two kinds of codon optimization strategies, respectively. Moreover, compared with the production of soluble pFSH and SUMO-pFSH, the production of soluble protein HSA-pFSH was significantly improved. Furthermore, the optimum pH and methanol concentration for expressing soluble HSA-pFSH in strain H3-3 were determined as 5.0-6.0 and 1.5-2% in shake-flask, and the yield of soluble HSA-pFSH could reach 40.8mg/l after purification. In vitro bioactivity assays showed that recombinant HSA-pFSH could efficiently stimulate cAMP synthesis in HEK293 cells expressing porcine FSHR. In conclusion, our results demonstrated that the application of phenotypic selection of aox1 mutants, combined with codon optimization, the choice of fusion partners, and fermentation optimization, considerably increased the yield of pFSH in supernatant of P. pastoris and thus provided a valuable reference for the large-scale recombinant expression of pFSH.