Abstract: 【Objective】The preliminary study on the protoplast formation of L.b is studied in this text;【Method】L.b is isolated and identification from the Yogurt and the biological characteristics was observed; the protoplast formation is studied with different concentration and treating time of mutanolysin;【Results】L.b is a slim rod ,gram-positive bacteria; The curves show that the growth lag phase is 2-16h; the protoplast formation rate of adding glycin（1mg/ml） was significance higer than the control; the protoplast formation of L.b was increased following the increasing dose of mutanolysin and time;【Conclusion】the optimum concentration and treating time of mutanolysin was 4ug/ml, 45min respectively.