The yeast fusant ZFF-28, which is high in biomass production and rich in selenium, was constructed after mutagenesis and protoplasts fusion between yeast strains. The total selenium content of ZFF-28 is 1.8 and 1.0 times higher than that of the parental strains Saccharomyces cerevisiae ZY-67 and Saccharomyces kluyveri SZY-198 respectively. Using single factor tests and a L16(4(3) x 2(1)) orthogonal design, the cultivation conditions was optimized as: 50mL culture in 250mL shake flasks in molasses containing 6% sugar and 60microg/mL Se at 28 degree C for 25h at 220 r/min, with the initial pH adjusted to 6.0 - 6.5. Under the optimized conditions, the biomass (dry weight) reached 8.2g/L and the Se content of the cells reached 2050microg/g, with organic and inorganic Se contents being 91% and 9% respectively.