Objective To explore the method of improving the isolation efficiency of influenza virus according to the comparative study between MDCK cell method and chicken embryo one in the isolation of influenza B virus. Methods A total of 42 respiratory specimens of influenza B positive cases detected by nucleic acid test from sentinel hospital of Huainan were inoculated into MDCK cells and chicken embryos respectively. The hemagglutination titer of the harvested strains (all of the first generation strains) and the type of the strains were determined by erythrocyte agglutination test and hemagglutination inhibition test. Results 23 strains of influenza B virus were isolated by MDCK cell method and 14 strains were isolated by chicken embryo method. The isolation rates were 54.76% and 33.33% respectively. 82.60% (19/23) and 71.43% (10/14) of the corresponding strains had hemagglutination titers of 1:8 and above,respectively.Compared with the chicken embryo method,the cell method was more sensitive,and the method of culturing chicken embryo with negative hemagglutination by cell method was negative,both of them were consistent. Conclusion Both MDCK cell method and chicken embryo one can be used for the isolation of influenza B virus. The combination of the two methods can improve the isolation efficiency of influenza virus.