Cultured meat technology is a novel and promising alternative strategy for meat production, and it provides an efficient, safe, and sustainable way to supply animal protein. Cytokines play an important role in promoting the rapid proliferation of cells, but the high cost and potential food safety concerns of commercial cytokines have hindered their application in large-scale cultured meat production. Herein, Saccharomyces cerevisiae C800 was used as a starting strain in which four cytokines were exogenously expressed simultaneously using the Cre-loxP system, including long-chain human insulin growth factor-1, platelet-derived growth factor-BB, basic fibroblast growth factor, and epidermal growth factor. Through promoter optimization, endogenous protease knockout, genomic co-expression, expression frame gene order optimization, and fermentation optimization, a recombinant strain CPK2B2 co-expressing four cytokines was obtained with a yield of 18.35 mg/L. After cell lysis and filter sterilization, the CPK2B2 lysate was directly added to the culture medium of porcine muscle satellite cells (MuSCs). CPK2B2 lysate promoted the growth of MuSCs and increased the proportion of G2/S cells and EdU+ cells significantly, indicating its efficacy in promoting cell proliferation. This study provides a simple and costsaving strategy by using S. cerevisiae to produce a recombinant cytokine combination for cultured meat production.