In this paper, the microorganism producing pyridoxal phosphokinases was isolated and applied to phosphorylate pyridoxal to pyridoxal phosphate with pyrophosphate acid. The product was detected by HPLC at 388 nm, and its retention time was at 1.75 min. C2-300 as the high productivity strain was isolated and its pyridoxal phosphokinase content reached 64 μg / mL.