To improve yeast strains for bioethanol production, yeasts with ethanol tolerance, thermotolerance, and ${eta}$-1,3-glucanase activity were bred using yeast genome shuffling. Saccharomyces cerevisiae $BY4742{Delta}exg1$/pAInu-exgA, which has extracellular ${eta}$-1,3-glucanase activity, and the Aspergillus oryzae and S. cerevisiae YKY020 strains, which exhibit ethanol tolerance and thermotolerance, were fused by yeast protoplast fusion. Following cell fusion, four candidate cells (No. 3, 9, 11, and 12 strains) showing thermotolerance at $40^{circ}C$ were selected, and their ethanol tolerance (7% ethanol concentration) and ${eta}$-1,3-glucanase activity were subsequently analyzed. All the phenotypes of the two parent cells were simultaneously expressed in one (No. 11) of the four candidate cells, and this strain was called BYK-F11. The BYK-F11 fused cell showed enhanced cell growth, ethanol tolerance, ${eta}$-1,3-glucanase activity, and ethanol productivity compared with the $BY4742{Delta}exg1$/pAInu-exgA and YKY020 strains. The results prove that a new yeast strain with different characters and the same mating type can be easily bred by protoplast fusion of yeasts.