Abstract10‐Hydroxy‐2‐decenoic acid (10‐HDA) is a terminal hydroxylated medium‐chain α,β‐unsaturated carboxylic acid that performs various unique physiological activities and has a wide market value. Therefore, development of an environmentally friendly, safe, and high‐efficiency route to synthesize 10‐HDA is required. Here, the β‐oxidation pathway of Escherichia coli was modified and a P450 terminal hydroxylase (CYP153A33‐CPRBM3) was rationally designed to synthesize 10‐HDA using decanoic acid as a substrate via two‐step whole‐cell catalysis. Different homologues of FadDs, FadEs, and YdiIs were analyzed in the first step of the conversion of decanoic acid to trans‐ ‐2‐ decenoic acid. In the second step, CYP153A33 (M228L)‐CPRBM3 efficiently catalyzed the conversion of trans‐ ‐2‐ decenoic acid to 10‐HDA. Finally, 217 mg L−1 10‐HDA was obtained with 500 mg L−1 decanoic acid. This study provides a strategy for biosynthesis of 10‐HDA and other α, β‐unsaturated carboxylic acid derivatives from specific fatty acids.