[Background] Studies have shown that the NADH oxidase (NOX) of Mycoplasma not only functions as an enzyme in cytoplasm, but also exists on the cell membrane as an adhesin. [Objective] To study the enzymatic activity and subcellular localization of the NOX of Mycoplasma synoviae (MS), analyze its potential role in the MS pathogenesis. [Methods] The recombinant MS NOX (rMSNOX) protein was prokaryotic expressed and purified. Then the enzymatic activity of purified rMSNOX and the factors affecting enzymatic activity were studied. In addition, the enzymatic specific activity, the maximum reaction rate (Vmax) and the Michaelis constant (Km) of the rMSNOX were determined. The MS positive chicken serum was used for Western blotting analysis of the immunogenicity of the rMSNOX. The Mycoplasma whole-cell, membrane and cytoplasmic fractions were prepared for Western blotting analysis to determine the subcellular localization of MSNOX using the rabbit anti-rMSNOX serum. [Results] The rMSNOX protein was successfully expressed in E. coli BL21(DE3) and purified. The relative molecular mass was about 53 kD. The enzymatic specific activity of the rMSNOX protein was determined as 14.17 IU/mg, the optimum enzymatic temperature was 37 °C, and the optimum pH was 7.5 by an enzyme activity analysis. The Km (NADH) of rMSNOX was determined as 244.0 μmol/L, and the Vmax as 21.8 μmol/(L·min) using the double-reciprocal method. The rMSNOX presented a specific binding to MS positive chicken serum, which proved that it had good immunogenicity. Subcellular localization analysis indicates that NOX protein distributed in MS cytoplasm and cell membrane. [Conclusion] This study demonstrates for the first time that MSNOX not only possesses NADH oxidase activity, but also is an immunogenic membrane protein, which provides a molecular basis for further exploring the role of NOX in the MS pathogenesis.