Dyeing wastewater is very hard to treat, and adsorption could be a good choice. Spent substrate of Pleurotus eryngii (SSPE) was first used to adsorb malachite green, safranine T and methylene blue from aqueous solutions, and the corresponding adsorption isotherm, thermodynamics and dynamics models were simulated. More than 93% of the dyes were removed with solutions with 100 mg/L of initial dye concentration, 1 g of SSPE and pH of 6.0 after adsorption for 4 h. Freundlich isotherm models fit better the adsorption data than Langmuir models. Adsorption of the dyes onto SSPE was a spontaneous exothermic process based on an adsorption thermodynamics model. SSPE could adsorb the dyes rapidly, and a second-order kinetics model fit better with the adsorption data than a pseudo first-order kinetics model. Accordingly, SSPE could be a good bio-adsorbent for the removal of malachite green, safranine T and methylene blue from the aqueous solution.