Abstract: The optimum conditions for extraction and separation of water-soluble nucleotide from fruit body of an edible fungus-Agrocybe cylindracea was studied. The crude nucleotides were assisted extraction with the grind bead . Three factors (grind time, ratio of liquid to solid, and loading capacity of grind beads) were investigated in the single-factor experiments. The process parameters were optimized by response surface analysis method. A multiple regression equation which was identified significant by regression analysis and the optimum extraction conditions were also determined (grind time: 12min, ratio of liquid to solid: 51:1 mL/g, loading capacity of grind beads: 12.5%). The theoretical yield for nucleotide was 7.02%. Furthermore, the actual yield was 6.94%, which was in accordance with theoretical result and thereby illustrated the fitted multiple regression equation was credible. Compared with traditional methods, grind bead assisted extraction not only produced high yield of nucleotides, but also greatly shortened processing time, which provided an industrial reference for developing new processes of nucleotides production from edible fungi.