Mycelial growth rate and biological efficiency of fruit body from ‘Xing 1’, ‘Xing 2’ and ‘Z xing’, three different Pleurotus eryngii varieties, were determined by different spent substrates. The results showed that the optimal conditions for Pleurotus eryngii cultivation were at temperature 24 ℃, substrate with 65% medium moisture and pH 6.0; Adding 40%–50% of spent substrate into different basic substrates, including cottonseed hulls, ramie byproduct and ramie root mediums could reach higher biological efficiency with 67.59%, 70.24% and 71.02%, respectively. The biological efficiency of ‘Xing 1’was higher than that of the other two varieties, but there was no significant difference. The results indicated that the spent substrate was suitable for Pleurotus eryngii cultivation.