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To remove heavy metals from aqueous solution and reclaim valuable materials from mushroom byproducts, Flammulina velutipes spent substrate (FVSS) was developed as a novel biosorbent for copper ion removal. Batch experiments demonstrated that ion removal was pH-, biosorbent dosage- and initial metal concentration dependent. The maximum removal capacity of 15.56 mg/g was achieved at pH 6.0 with a biomass dosage of 3.0 g/L and initial copper ion concentration of 50 mg/L. The adsorption data were in compliance with the Langmuir isotherm and a pseudo-second-order kinetic model. Thermodynamic studies revealed the biosorption process was endothermic, random, and spontaneous. FT-IR spectral analysis confirmed that hydroxyl, amino, carbonyl, and phosphate groups on the biosorbent surface were involved in the biosorption. The uneven surface and porous structure of the biosorbent was propitious for quickly capturing the metal ions from aqueous solution. EDX spectra revealed that the copper ions were loaded on the surface of the biosorbent. XRD patterns showed the formation of copper-containing compounds.