Abstract: After enlarged culture of Cordyceps militaris Link and liquid fermentation,centrifugal separation was carried out to obtain mycelial liquid.The conditions of protoplast preparation were optimized from four aspects:osmotic pressure stabilizer,lysozyme,lysis time and bacterial age.The results showed that the optimal osmotic pressure stabilizer for protoplast preparation was 0.6 mol/L sulfatase,and the optimal osmotic pressure stabilizer was 0.6 mol/L sulfatase.While the optimum enzyme solution was 1% cellulase,the optimum bacterial age was 5 days,and the optimum cracking time was 2 hours.The orthogonal experiment of L₂₀(5⁴) showed that 1.46×10⁷ protoplasts could be prepared under the optimum conditions.The results of ultraviolet mutagenesis experiment showed that the irradiation dose was about 40 seconds,and the mutagenesis distance was inversely proportional to the lethality.When the total time of mutagenesis was the same,the number of mutation was less affected,and the effects of multiple and single treatment were similar.The polysaccharide content was then used as an indicator to screen the mutagenized strains,and we successfully got the mutants that showed higher content of the polysaccharide than that of common strains.The polysaccharide content of 17^# was 6.01%.The stability test of the screened strains showed that the ability of polysaccharide production of screened strains decreased less after three subcultures and the screened strains had better stability.