To improve the production of L-valine, the He-Ne laser is used for Brevibacterium flavum to induce mutagenesis. The He-Ne laser with different power and duration is tested, and the best laser treatment condition that the intensity and time of irradiation is 15 mW and 20 min, respectively is decided. The positive mutants are selected directionally and stepwise on the agar plates containing extra L-valine of different concentrations, and L-valine overproducing mutant is derived with production increased by 25.50%. The results show that the mutant strain can keep steady properties for at least 10 generations. He-Ne laser is an effective technique for microbial mutagenesis, and the He-Ne laser with low power is suitable for Brevibacterium flavum mutagenesis.