Abstract: In order to select a new strain of Cordyceps militaris that could synthesize cordycepin and accumulate oil efficiently, the protoplast fusion was performed on the strain FY1421 of Cordyceps militaris with high yield of cordycepin and the strain Z0108 of mortierella isabellina with high efficiency in oil synthesis, especially in unsaturated fatty acid synthesis. The conditions of protoplast formation and regeneration, inactivation and fusion of parents were screened and optimized. And the fusions were obtained by combining the initial screening of 96 microwell plate with the shaking flask fermentation. The results showed that the strain FY1421 of Cordyceps militaris was treated with a mixture of 1.5% lysozyme+0.5% snail enzyme+0.5% cellulase for 120 min, with the largest number of protoplasts and the highest regeneration rate. The strain Z0108 of mortierella isabellina was treated with a mixture of 1% lymolytic enzyme+1% snail enzyme+1% cellulase for 4 h, with the largest number of protoplasts and the highest regeneration rate. The protoplast of FY1421 was inactivated by 60℃ water bath for 10 min, and the protoplast of Z0108 was inactivated by ultraviolet ray for 30 s, both of which could achieve 100% inactivation rate. And the inactivated parents were fused with 40% PEG for 10 min at 35℃which could achieve the highest fusion efficiency. The fusion strain RCS262 was screened from 650 strains of fusions, and the content of cordycepin with the shaking flask fermentation reached 5926 μg·g^-1, while the oil content reached 37.5%. The protoplast fusion technique was used to conduct the interspecific hybridization of Cordyceps militaris and Mortierella isabellina, and the fusion strain obtained successfully could not only produce high yield of cordycepin but also accumulate oil efficiently.