10-hydroxy-2-decenoic acid (10-HDA), a unique unsaturated fatty acid present in royal jelly, has attracted considerable interest due to its potential medical applications. However, its low concentration in royal jelly and complex conformational structure present challenges for large-scale production. In this study, we designed and constructed a de novo biosynthetic pathway for 10-HDA in Escherichia coli. Initially, we introduced the heterologous thioesterase UaFatB1 to hydrolyze trans-2-decenoyl ACP to produce trans-2-decenoic acid, a key precursor for 10-HDA. Subsequently, we employed the bacterial cytochrome P450 enzyme CYP153AMaq to catalyze the terminal hydroxylation of trans-2-decenoic acid. Furthermore, through redox partner engineering and directed evolution, we identified the optimal combination for 10-HDA production: CYP153AMaq Q129R/V141L mutant with redox partner FdR0978/Fdx0338. Finally, we optimized the fermentation conditions and achieved a 10-HDA titer of 18.8 mg/L using glucose as primary carbon source. Our work establishes a platform for producing α,β-unsaturated fatty acids and their derivatives, facilitating further studies on these compounds.