Abstract: The influence of cane molasses on the biosynthesis of coenzyme Q₁₀ produced by Rhodobacter sphaeroides was investigated. The cane molasses adding process was optimized, which improved the sugar conversion rate and greatly reduced the production cost. The results showed that different carbon source substrates had a great influence on the synthesis of coenzyme Q₁₀ products, and glucose was the most suitable carbon source, which was beneficial to the growth of Rhodobacter sphaeroides and the synthesis of coenzyme Q₁₀. Through orthogonal experiments, the fermentation medium of coenzyme Q₁₀ was optimized. When the mass concentrations of sugarcane molasses, glucose and KH₂PO₄ were 20 g/L, 30 g/L and 1 g/L, respectively, the growth and coenzyme Q₁₀ synthesis of erythroid bacteria were favorable, and the fermentation titer of coenzyme Q₁₀ reached 380.1 mg/L. The sugar conversion rate was 12.71 mg/g. The fermentation was amplified on a 30 L fermenter, and the physiological parameters of coenzyme Q₁₀ fermentation were collected and analyzed, which further verified that sugarcane molasses was conducive to the maintenance of the metabolic activity of thalli, and thus promoted the biosynthesis of coenzyme Q₁₀.