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Yarrowia lipolytica is an oleaginous yeast species with the ability to grow on a number of substrates types, especially industrial wastes. This paper concerns the statistical optimization of fermentation parameters and media to ensure consistent and improved Y. lipolytica protein production. A strain of Y. lipolytica A-101 was observed to be proficient in producing single cell protein, amino acids, and vitamin B12 while utilizing biofuel waste instead of a complete YPD medium for yeast growth. A fractional fractal design experiment was then applied, and the two fermentation parameters of temperature and pH were recognized to have a significant effect on the protein and amino acid production. Subsequently, the response surface methodology with a three-level complete factorial design was employed to optimize these influential parameters. Therefore, five different measuring systems were utilized to construct a quadratic model and a second-order polynomial equation. Optimal levels of parameters were then obtained by analysis of the model and the numerical optimization method. When the Y. lipolytica A-101 was cultivated at optimized pH (5.0) using biofuel waste as a medium, the protein concentration was increased to 8.28—a 44% enhancement as compared to the original (3.65). This study has thus demonstrated a beneficial way to cultivate Y. lipolytica A-101 on biofuel waste for enhanced production of single cell protein and amino acids for use in human diet and in animal feed.