Phellinus igniarius is a medicinal fungus that utilizes lignin as a nutrient substrate. This fungus has a weak lignin degradation ability and, as a result, a slow growth rate. Laccases are crucial enzymes for lignin degradation in P. igniarius, and thus, the cultivation of strains with high laccase activity is expected to increase the growth rate of P. igniarius. To generate P. igniarius strains with high laccase activity, we performed laser mutagenesis of P. igniarius protoplasts and screened for mutants with high laccase activity. Our results showed that the laser power density and P. igniarius protoplast survival rate exhibited a power-function relationship. The power density threshold value between lethality and growth promotion was 0.24 mW/mm(2). Mutagenesis was carried out using a laser beam diameter of 3 mm and an irradiation period of 40 min. After five generations of selection, we identified a high laccase activity strain, termed SJZ2. The laccase activity in SJZ2 during 4 h of fermentation was increased by 36.84% in comparison with the control and ranged from 0.20216 to 0.27664 U. The Km and Vmax of the laccase produced by SJZ2 were 0.21 mmol/mL and 0.53 mmol/L/min, respectively. This study demonstrated the feasibility of laser mutagenesis of P. igniarius protoplasts for the selection of high laccase activity. This study characterized the key factors in the laser mutagenesis process of P. igniarius protoplasts and provided a reference for the application of lasers in biological mutagenesis. Future studies should evaluate the bioactive functionality and stability of this novel strain of P. igniarius, particularly the organoleptic and medical characteristics of the fruiting bodies.