Terminal ω-hydroxy fatty acids are important industrial precursors for the synthesis of polyhydroxy fatty acids, nylons, and lactones. The cytochrome P450 monooxygenases are able to catalyze hydroxylation of fatty acids for synthesis of terminal ω-hydroxy fatty acids. Directed evolution is the promising strategy to engineer the P450 and shift its regioselectivity towards the terminal position. In this article, a high throughput screening method to detect terminal ω-hydroxy fatty acids for identifying P450 variants with selectivity for terminal oxidation was developed and validated. A ω-hydroxy fatty acids specific fatty alcohol oxidase (FAO) from Candida tropicalis was applied to quantitatively detect the products. The formation of hydrogen peroxide during the process of oxidation of ω-hydroxy fatty acids is coupled with horse radish peroxidase (HRP) for color reaction with the chromogenic substrate of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; ABTS) in 96 multi-well plates. The FAO-ABTS system was successfully applied to the detect the P450 BM3 mutant capable of catalyzing the terminal ω-hydroxylation of pentadecanoic acid and palmitic acid.