© 2019 Elsevier B.V.In this study, a multi-step experimental design of Response Surface Methodology (RSM) was applied to optimize the medium conditions for the maximum removal of Methylene Blue (MB) from aqueous solution by a novel fungi A. campestris as a biosorbent. In first step, the effect of factors (initial dye concentration, temperature, contact time, pH, agitation speed and adsorbent dosage) was obtained using Plackett Burman Design (PBD). Then Steepest Ascent (SAD) used to predict the optimum region of effective factors in the second step. Central Composite Design (CCD) was utilized to evaluate the optimum medium conditions of effective parameters for the removal of cationic dye on last step. RSM indicated that optimum conditions of initial dye concentration, agitation speed and medium temperature for maximum removal of MB (95%) were achieved as 130.90 mg L−1, 125 rpm and 41.87 °C, respectively. The activation energy (Ea) was determined as 149.1, -178.6, 154.5 and 382.3 kJ/mol for 20, 50, 100 and 200 mgL−1 respectively. The characterization of adsorption process was confirmed by Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). Adsorption isotherm was used to describe the adsorption equilibrium studies at different temperatures. Langmuir isotherm shows better fit than Freundlich and Temkin isotherms. Thermodynamic parameters like the enthalpy 15 kJ/mol (ΔHo), entropy 66.59 J/molK (ΔSo) and Gibbs free energy -4.47 kJ/mol (ΔGo) were evaluated and also, ΔGo shows a negative values indicating that the adsorption process was spontaneous and endothermic in nature. The results show that a multi-step statistical optimization designs is successful applied to experiments and novel and endemic biomass of Agaricus Campestris is an appropriate biosorbent and has a specific affinity for removal of Methylene Blue at under optimal conditions.