The invention provides a preparation and regeneration method of pythium oligandrum protoplasts. The reparation and regeneration method comprises the following steps: a, preparing thallospore hybrids; b, preparing protoplasts; c, purifying the protoplasts; and d, regenerating the protoplasts. According to the method, a complex enzyme system containing lysing enzyme, cellulsase and lyticase is adopted for carrying out enzymolysis on the mycelia of pythium oligandrum, then the pythium oligandrum protoplasts are obtained, and the protoplasts are regenerated through liquid shaking culture adopting a regeneration culture medium taking mannitol as an osmotic pressure stabilizer. The number of the pythium oligandrum protoplasts prepared by adopting the method is large, the yield is high, and the activity is good; through the liquid culture regeneration path, relatively high regeneration rate can be obtained, and the regeneration rate can achievce 1.25%. The method is easy and convenient to operate and good in stability, the obtained pythium oligandrum protoplasts can completely satisfy the research and application such as late gene genetic transformation and cell fusion. The invention further provides composite enzymatic hydrolysate for preparing the pythium oligandrum protoplasts, the composite enzymatic hydrolysate is a key factor in the preparation process of the pythium oligandrum protoplasts, and the enzymatic hydrolysate is suitable for being further developed and applied.