Abstract Background Citrus peel waste (CPW) is a pectin‐rich agro‐industrial residue with potential as a renewable substrate for mucic acid production. However, commonly used laboratory strains of Saccharomyces cerevisiae require nutrient supplementation, which limits their industrial applicability. This study aimed to restore prototrophy, enhance fermentation performance, and optimize process conditions for efficient mucic acid production from CPW. Results Prototrophic derivatives were engineered, enabling nitrogen‐independent growth. Compared with the parental strain, the prototrophic strain achieved nearly 30% higher mucic acid titers and improved co‐utilization of D‐galacturonic acid with xylose. In simultaneous saccharification and fermentation of CPW, mucic acid production increased by 23% under nitrogen‐limited conditions. Response surface methodology identified optimal conditions of 5% CPW, 7.89 g DCW/L inoculum, 130 rpm agitation, and 48 h incubation, yielding 15.38% mucic acid, closely matching the predicted maximum. Conclusion By combining strain engineering, high‐cell‐density cultivation, and optimization, this study establishes a promising platform for the valorization of agro‐industrial residues into high‐value biochemicals, with potential benefits for cost‐effectiveness and sustainability. © 2025 Society of Chemical Industry (SCI).