The adsorption of Rhodamine-B (Rh-B) from aqueous solutions onto spent mushroom substrate (SMS) and mushroom substrates prior to use (MSP) were comparatively studied in terms of the adsorption parameter effects, isotherms, and kinetics. It was found that an acidic pH of the dye solution was detrimental for basic Rh-B dye adsorption, which favored the electrostatic attraction between the cationic Rh-B and negative SMS and MSP. The adsorption isotherms of Rh-B onto the SMS and MSP followed the Langmuir model rather than the Freundlich or Temkin models. The fitted adsorption capacity of the SMS (107.5 mg/g) was approximately double that of the MSP (47.6 mg/g), which indicated a higher surface area and the presence of more adsorption functional sites that were created during edible fungus (Grifola frondosa) cultivation. Moreover, the experimental adsorption data of the SMS and MSP obeyed pseudo-second-order kinetics. The intraparticle diffusion plots revealed the multilinear adsorption nature of the SMS, which included boundary layer adsorption, intraparticle diffusion, and pore diffusion. However, only intraparticle diffusion played a major role in the adsorption of Rh-B onto MSP. It was concluded that the utilization of agricultural waste for edible fungus cultivation would not only improve the value of the agricultural waste itself, but can also remove Rh-B from aqueous solutions.