Maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) are three staple crops and accordingly it is very meaningful to optimize the condition of their protoplasts isolation. The concentration of the enzyme, the time of isolation and centrifugal force in protoplast isolation were investigated to find their effects on protoplast yield and viability using leaves of maize (Zong 3), wheat (Chinese Spring) and rice (Nipponbare). The results show that the concentration of the enzyme and the time of isolation affected the protoplast yield significantly. Although the yield of protoplast was increased with high concentration of enzyme and long incubated time, it led to too much cells breakdown. The orthogonal experimental design results show that the best condition of maize protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 7 h, 100×g 2 min and the protoplasts yield was 7×106 cells/g fresh weight (FW); the best condition of wheat protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 5 h, 100×g 2?min and the protoplasts yield was 6×106 cells/g FW; the best condition of rice protoplast isolation was Cellulase R-10 2.0%, Macerozyme R-10 0.7%, 50 r/min 7 h, 1 000×g 2 min and the protoplasts yield was 6×106 cells/g FW. The vitalities were more than 90% using fluorescein diacetate staining method. 50%?80% transformation efficiency was obtained when protoplasts were transformed by green fluorescent protein using PEG-Ca2+ method.