[Objective] There is almost no report about the sheep-derived Clostridium butyricum in the world at present. It is the study that optimized of liquid fermentation for sheep-derived Clostridium butyricum HDRyYB1. This study sets the foundation for Clostridium butyricum HDRyYB1 industrial product as feed additive in livestock. [Methods] We used the Plackett-Burman (PB) design and response surface methodology (RSM) though determing the spore number of Clostridium butyricum HDRyYB1 in this report. [Results] The results showed that the concentration of wheat flour, rice flour, fish meal had remarkable effect on the spore number. The optimum medium for incubating the Clostridium butyricum HDRyYB1 was composed of 3.72% wheat flour (w/v), 0.90% fish meal (w/v), 3.96% rice flour (w/v), 0.60% yeast powder (w/v), 0.19% NaCl (w/v), 0.19% MgSO4·7H2O (w/v), 0.01% KH2PO4 (w/v), 0.01% NaHCO3 (w/v), 0.48% CaCO3 (w/v), at 37 °C, pH 7.2?7.4, bottled in 100/250, inoculated quantity 3%. Under this condition, the spore number was 1.478×108 colony-forming units (CFU)/mL after fermented completely in 18 h, which was 2.7 times than before. [Conclusion] These results indicate that the fermentation medium has been optimized, it can be used in the expanded fermentation experiment to verify its application value in production practice.