Coenzyme Q₁₀ (CoQ₁₀ ) is an endogenous enzyme cofactor that may be found in every cell in the human body and an important natural antioxidant widely used in many fields, such as medicine, food and cosmetics. The protoplast fusion is an important process in the genome shuffling. In order to improve the efficiency of the genome shuffling and the CoQ₁₀ production, this paper studies the conditions of the protoplast preparation and the regeneration of CoQ₁₀ to produce the strain Rhodobacter sphaeroides. The parameters of the proper protoplast formation and the regeneration were determined through the growth curve and the orthogonal experiment: the lysozyme concentration is 1mg/ml, the enzymatic temperature is 37℃; the enzymatic time is 1h and the sucrose concentration is 10%, and under these conditions, the protoplast formation rate is 96.1% and the protoplast regeneration rate reaches 28.8%. Therefore, the optimized conditions of the protoplast preparation and the regeneration for the genome shuffling of Rhodobacter sphaeroides are obtained.