Acetyl-CoA serves as foundational precursor and energy source for various biosynthesis pathways. The insufficient supply of acetyl-CoA in cytosol is usually a bottleneck for exogenous chemical synthesis in engineered microbes such as yeast. Exportation of acetyl-CoA from peroxisomes, an exclusive organelle for fatty acids β-oxidation, may be an effective way to solve this problem. In this study, we developed a peroxin (PEX) engineering strategy to modulate peroxisome assembly in S. cerevisiae. Then, robust peroxisomes were constructed with improved acetyl-CoA supply by up to 98%, which further led to the increased liquiritigenin titer (1102.41 mg/L), the highest reported value so far. We also demonstrated that the PEX–mediated peroxisome engineering strategy can be extended across yeast species. Hybrid peroxisomes with tailored function were constructed in S. cerevisiae by transplanting selected PEXs from Y. lipolytica. Our study provides novel mechanistic insights into the “PEXs–peroxisome assembly–acetyl-CoA synthesis” relationship.