In order to realize the industrial production of glyoxylic acid, the expression of glycine oxidase (ThiO) was optimized using genetic engineering technology. The soluble expression of recombinant strains was verified by SDS-PAGE electrophoresis. The ability of recombinant bacteria to produce glyoxylic acid was tested by whole cell transformation. With the optimization of reaction conditions, its applicability in the enlarged transformation system was investigated. The strain Bacillus subtilis 168 (pP43NMK-ThiO) achieved intracellular soluble expression and the yield of glyoxylic acid from glycine by the whole cell transformation surpassed the highest yield of glyoxylic acid produced by current biological methods. The best reaction conditions were 30 ℃ and pH value 8.3. The whole cell catalyst maintained high thermal stability at 35 ℃, and more than still maintains more than reaction activity after 8 h incubation. Under the optimal reaction conditions, the yield was further increased to 30% by adding catalase at a final concentration of 1%. The results from the 3 L fermentation tank are consistent with those obtained from the shake flask, showing stable conversion capacity. The results lays the foundation for the industrialization of the preparation of glyoxylic acid by the biological method.