L‒Glutamate oxidase (LGOX) from Streptomyces sp. X‒119‒6 has strict substrate specificity towardL‒glutamate. Recently, we solved the X‒ray crystal structure of LGOX and this revealed that Arg305 inthe active site is the key residue involved in substrate recognition. Therefore, we created 19 mutantenzymes of R305X‒LGOX by saturation mutagenesis. One of them R305D‒LGOX, Arg305 substitutedwith Asp exhibited oxidase activity for L‒Arg. Optimum pH of R305D‒LGOX mutant enzyme was pH8.5. Interestingly, the activity of R305D‒LGOX toward L‒Arg was inhibited by phosphate. And furthermore,the substrate specificity of R305D‒LGOX was affected by using buffer. The results of inhibitionanalysis suggest, that phosphate is a competitive inhibitor of R305D‒LGOX when L‒Arg is used assubstrate. Kinetic analysis of R305D‒LGOX showed that Km value and kcat value of R305D‒LGOX towardl-Arg were 0.68 mM and 6.7 s-1 respectively. In this study, we showed that R305D‒LGOX mutantenzyme is a novel l-arginine oxidase and useful for l-arginine biosensor.