Long-chain hydroxy fatty acids (HFAs) are rare in nature but have many promising industrial applications. In this study, we developed a biosynthesis method to produce long-chain ω-hydroxy fatty acids. Through disruption of the acyl-CoA synthetases FAA1 and FAA4 and the fatty acyl-CoA oxidase POX1, a Saccharomyces cerevisiae strain was engineered to accumulate free fatty acids (FFAs). Subsequently, the cytochrome P450 monooxygenase CYP52M1 from Starmerella bombicola was introduced to convert FFAs to HFAs, leading to the production of C16 and C18 HFAs at the ω or ω-1 positions. Next, CYP52M1 was reconstituted with the homologous reductase S. bombicola CPR and the heterologous reductase Arabidopsis thaliana cytochrome P450 reductase. The results showed that the CYP52M1-AtCPR1 system significantly increased the hydroxylation in FFA. Moreover, a self-sufficient P450 enzyme system was constructed to achieve higher transformation efficiency. Finally, fed-batch fermentation yielded as much as 347 ± 9.2 mg/L ω-HFAs.