Abstract: The six kinds of traditional Chinese medicine granule residues were used to optimize the formula of the culture substrate for Ganoderma lucidum. The optimal substrate formula of traditional Chinese medicine residue for Ganoderma lucidum was selected by taking the economic character of fruiting body and the quality of Ganoderma lucidum (the content of polysaccharide and triterpenes) as the indexes. Meanwhile, three fruiting bodies were selected to prepare the strains by using the method of tissue isolation, in order to investigate the feasibility and reproducibility of the operation. The results showed that the 6 kinds of traditional Chinese medicine residues could be used as the culture substrate for Ganoderma lucidum, and the shape, yield and quality of the fruiting bodies of Ganoderma lucidum cultured with the formula 3 (added residue of Ligusticum chuanxiong) were the best. The polysaccharides content of 2.16% and the content triterpenes of 1.03% were respectively 1.35 and 1.49 times higher than those of the formula 1 (CK, not added Chinese medicine residue). The strain preparation by using the method of tissue isolation was simple and reproducible. The strains could be isolated from Ganoderma lucidum in about 9 days after the simple isolation for 1-3 times.