To solve the problems of heavy metal pollution and agricultural wastes reclamation, spent substrate of pleurotus oyster was used as adsorbents to remove Cu2+ from aqueous solution. The effects of pH value, adsorption time, temperature and initial Cu2+ concentration on the adsorption behavior were determined by single factor experiments. The mechanisms were preliminarily investigated by SEM-EDX, FTIR and XRD analysis. The results of single factor experiments showed that the adsorption rate and capacity reached 74.46% and 0.7446 mg x g(-1) respectively at an adsorbent concentration of 10 g x L(-1), a pH of 6, an adsorption time of 120 min, an adsorption temperature of 30 degrees C and an initial Cu2+ concentration of 8 mg x L(-1). The experimental data fitted well with Langmuir isotherm models and R2 reached 0.994 9, indicating the adsorption was a monolayer chemisorption. SEM-EDX, FTIR and XRD analysis indicated that the adsorption process mainly depended on the physical and chemical adsorption onto the substrate surface through electrostatic attraction, complexation and coordination reaction. The -OH, -COOH and -NH were the functioning groups for Cu2+ adsorption.