To provide a visible material for the study of infection and control of fusarium root rot of panax, through observing the optimal spores germination time, the composition of lyases and the type of osmotic pressure stabilizer, the preparation and transformation technologies of ginseng root rot pathogen-Fusarium oxysporum′s protoplast were studied in the experiment. The results revealed that the optimal conditions were young hyphae obtained in YEPD cultured for 11 h, hydrolysis temperature 37 ℃, speed of the shaker 180 r/min, and osmotic stabilizer 0.7 mol/L NaCl. When hydrolysates containing 0.01 g/mL driselase, 0.03 g/mL lysozyme, 0.02 g/mL cellulose and 0.005 g/mL snailase were used to hydrolyze 0.25 g young hyphae, the hyphae harvested were the most favorable for the formation and release of protoplasts, with a total yield of 2.8×10⁷ protoplasts/mL. The exogenous DNA was successfully transferred to F. oxysporum by the PEG-mediated transformation and the transformation frequency of 0083 was 1 400 transformants per mg DNA, which showed hygromycin resistance and fluorescence signal. After six generations of subculture, the representative transformant FOG1 showed stable morphology, sporulation and fluorescence. In conclusion, PEG-mediated protoplast transformation is quite stable and efficient.