The present invention belongs to microbial technology field and relates to a strain producing toluene o-xylene monooxygenase (Arthrobacter woluwensis) HW-1 and its application in preparation of 5-methylpyrazine-2-carboxylic acid by microbial fermentation. The present invention providing a new strain HW-1 which could produce toluene o-xylene monooxygenase, and the strain is identified as Arthrobacter woluwensis. The strain is firstly found to convert 2, 5-dimethylpyrazine by bio-fermentation to obtain the medicine intermediate 5-methylpyrazine-2-carboxylic acid. The concentration of accumulated product could reach 34.19 g/L and the yield rate is 81.4% by shake flask fermentation. Compared with 20.41 g/L reported in the literature, this method has a greater advantage and could be industrialized. The conditions to prepare 5-methylpyrazine-2-carboxylic acid provided in the present invention is mild, the reaction process is controllable and has good performance in environmental protection and energy saving. So this invention has great value in industry.