【Objective】To breed Athelia rolfsii strain with high yield of polysaccharide and low yield of oxalic acid. 【Method】 The protoplast fusion technique was adopted with original strain AY6657741 and parental strains of high polysaccharide yield Ar-1 and Ar-2, which were screened from AY6657741 by NTG with UV. The orthogonal test was designed to obtain the optimal conditions for preparation of protoplast fusion strains.【Result and conclusion】 The Ar-1 and Ar-2 were suspended by 5.56 mmol·L^-1 β-mercaptoethanol. Osmotic pressure stabilizer was 0.4 mol·L^-1 KCl. Ar-1 and Ar-2 were hydrolyzed by 3.1 mg·mL^-1 compound enzyme (2% cellulase, 2% snailase, 4% lywallzyme) for 55 min at pH 5.5, under the 32 ℃ condition. Protoplast formation rate and regeneration rate were 93.534% and 18.921% respectively. A strain, named AY-3, with high polysaccharide yield and low oxalic acid yield was screened by protoplast fusion under the condition of 0.4 g·mL^-1 polyethylene glycol 6000 for 25 min at 30 ℃. Compared with Athelia Rolfsii AY6657741, the polysaccharide yield of strain AY-3 increased by 54.42%(24.673 g·L^-1), and oxalic acid yield reduced by 43.57%(0.281 g·L^-1).