A new assay method of ${alpha}-Oxidase$ (fatty acid : oxygen dioxygenase, 1-decarboxylating) was developed using a bioluminescence reaction system of marine luminous bacterium, Photobacterium phosphoreum. ${alpha}$-Oxidase was isolated from a cucumber (Cucumis sativus). Pentadecanoic acid was used as a substrate, and the product, tetradecanal, was analyzed with a bacterial luciferase-coupled reaction. Initial light intensity was directly related to the concentration of tetradecanal in the range of 1 nM to 10 ${mu}M$. Optimal pH and temperature were 7.5 and $25^{circ}C$, respectively. Optimal pentadecanoic acid concentration in a standard assay of ${alpha}$-oxidase was 0.1 mM. The Km value of pentedecanoic acid was $85{mu}M$. This method is straightforward, rapid, convenient, and easy. Its needs no treatment or extraction of reaction mixture.