|| Novel glycine oxidase (GlyOX) from Marinomonas mediterranea depends on cysteine tryptophilquinone (CTQ) and catalyzes the oxidative deamination of glycine to produce a glyoxylate, ammonia, and hydrogen peroxide. M. mediterranea GlyOX genes (goxA and goxB) were cloned and recombinant GlyOX was heterologously expressed by E. coli. The purification of recombinant GlyOX was carried out by metal affinity and DEAE-Toyopearl 650M column chromatographies. M. mediterranea GlyOX was homotetramic with a molecular mass of 76kDa and showed optimum activity around 30°C and at pH 5.0, and stability below 50°C and between pH 5.0 to 9.0. M. mediterranea GlyOX shows a strict substrate specificity toward glycine, and the Michaelis constant for glycine was 0.5mM. M. mediterranea GlyOX could determine the quantity of glycine in human serum and human blood plasma with high sensitivity. This study revealed the catalytic and structural properties of M. mediterranea GlyOX with high substrate specificity.