For the first time, polysaccharide synthase activity was used as the cut-in condition in this study and the fermentation conditions of Coprinus comatus intracellular polysaccharide (IPS) were further optimized. The optimal conditions were 23.65 g/L of carbon source, 47.44 g/L of nitrogen source, and pH 5.73. C. comatus IPS was obtained under the optimal conditions and its antioxidant properties were evaluated. Next, in vitro proliferation and Lactobacillus rhamnosus acidifying activity were assessed. IPS had good antioxidant properties and could promote the growth and acid production of probiotics. The antioxidant properties and probiotic proliferation and acidifying activity of IPS were improved to a certain extent under the optimal conditions. Thus, our findings contribute to establishing a novel fermentation process that can produce IPS with better performance. Meanwhile, IPS has great application potential in medicine.