Pleurotus ostreatusis one of the most widely cultivated edible fungi in China and has been facing some challenges, such as strain degeneration and yield reduction probably due to viral infections. Sixteen virus-like infected strains of Oyster mushroom were collected. Oyster mushroom spherical virus（OMSV） was detected in six P.ostreatus strains and Oyster mushroom isometric virus（OMIV） was not detected in all strains by RT-PCR using specific primers. The full-length sequence of coat protein gene of OMSV in Po6 strain which has the strongest amplification was determined. The comparison of the coat protein sequence showed 98.18% nucleic acid identity and 97.89% amino acid identity with OMSV Korean isolate（Genbank ID: NC＿004560.1）. After eliminating OMSV in Po6 strain by protoplast regeneration method, the mycelia growth rate and biomass increased obviously, and the phenotype was closer to the healthy ones. It indicated that OMSV probably inhibits mycelia growth and fruiting body development of P. ostreatus.