Currently, button mushroom, Agaricus bisporus is one of the most consumed mushroom in the world. However, despite of its importance in food market, molecular genetic modification method for breeding of A. bisporus is not well established. In this study, we optimized yield of A. bisporus protoplast with Lysing enzyme, Chimax-N and cellulase. With this composition, 1.0 × 108/mL of protoplasts were obtained reliably. PEG-mediated transformation with spermidine showed almost 100-fold higher yield than non-spermidine method.