In the industrialized production of mushrooms usually only one flush of fruitbody is harvested, so that nutrients and energy in the substrate is not fully exploited. In this study, the spent Pleurotus eryngiisubstrate was recycled for the cultivation of Agrocybe chaxinguunder ambient temperature. Six formulae were tested: (1) Control: 98% spent substrate, 1% sucrose, 1% lime; (2) Control + 10% wheat bran; (3) Control + 20% wheat bran; (4) Control + 10% T. molitorfeces; (5) Control + 20% T. molitorfeces; (6) Control + 10% wheat bran + 10% T. molitorfeces. Two flushes of fruitbody were harvested, the control substrate resulted in a biological efficiency of 40.42%; the formulae with supplementation of 10% wheat bran, 20% wheat bran and 10% T. molitorfeces significantly increased biological efficiency to 52.50, 54.61 and 51.56%, respectively, and supplementation of 20% T. molitorfeces, or 10% wheat bran plus 10% feces further significantly increased biological efficiency to 62.95 and 61.10%, respectively. All supplemented substrates had significantly higher cellulose and laccase activity than the Control (cellulase 0.10 U/g; laccase 41.00 U/g), which were 10% wheat bran (0.15 U/g; 72.67 U/g), 10% T. molitorfeces (0.17 U/g; 98.33 U/g), 20% wheat bran (0.22 U/g; 76.00 U/g), 20% T. molitorfeces (0.27 U/g; 87.00 U/g), 10% wheat bran plus 10% T. molitorfeces (0.25 U/g; 97.67 U/g), respectively. Spent Pleurotus eryngiisubstrate was promising for cultivation of Agrocybe chaxingu, especially when supplemented with 20% T. molitorfeces, or with 10% T. molitorfeces plus 10% wheat bran.