cis-13,16-docosadienoic acid (DDA) is an omega-6 polyunsaturated fatty acid with potential applications in food and health fields. Previously, DDA was successfully synthesized using Yarrowia lipolytica Po1f (Δku70). However, the limitations remain for improving its production. In this study, Yarrowia lipolytica Po1f (Δku70, ΔTrp) was engineered to successfully produce DDA through systematic metabolic engineering, including screening of heterologous Δ12-desaturases and Δ9-elongases, fusion of the Δ9-elongase with ELO1-elongase using optimal linkers, and copy number increase along with promoter replacement of EhELO1, and disruption of PEX10 to block the fatty acid consumption pathway. Finally, a DDA content of 0.47% of total fatty acids (TFAs) was achieved, with a titer of 5.34 mg/L in shake flasks, which represents the highest DDA titer reported to date at this scale without fermentation optimization. These results establish the sustainable microbial platform as a viable alternative to conventional DDA production methods.