Abstract: The effects of parameters including solution pH value, initial Cu²⁺ concentration, contact time, spent shiitake substrate dosage, and temperature on the adsorption of Cu²⁺ on the spent shiitake substrate were studied, and the adsorption mechanisms were also analyzed. As the initial pH value lowers, the adsorption capacity decreases. The adsorption of Cu²⁺ on the spent shiitake substrate follows well by the Langmuir and Freundlich equilibrium adsorption models. The maximum adsorption capacity of Cu²⁺ is 33.11 mg·g^-1. The equilibrium time is 1 h. The adsorption kinetic data fit the pseudo-second-order model very well with the correlation coefficient of 0.9995. The adsorbent dosage is optimized to 10 g·L^-1. Raising the temperature leads to an extreme drop of adsorption capacity. Thermodynamic studies indicate that the biosorption process is exothermic and can react spontaneously more easily at lower temperatures. By means of scanning electron microscopy and Zeta potentials, it is found that the adsorption of Cu²⁺ on the spent shiitake substrate is mainly a physical process at lower pH values, while a chemical one at higher pH values.