<p>This study presents the initial physiochemical characterization of Djulis (<i>Chenopodium formosanum</i>) spent mushroom substrate (SMS) biochar and its application for methylene blue (MB) dye adsorption. Biochar was produced over a wide temperature range; 800&#xa0;°C and 60-min retention time resulted in the best product having a specific surface area of 289.44 m²&#xa0;g⁻¹. Functional group analysis revealed the presence of hydroxyl (OH) and carboxyl (COOH) groups. MB adsorption was evaluated under different conditions: dosage, pH (3–11), and temperature. Increased biochar dosage and higher temperatures improved MB removal efficiency. Zeta potential (<i>ζ</i>) values (− 11.43 to − 45.6&#xa0;mV) indicated stable suspension. The pseudo-first-order (PFO) and pseudo-second-order (PSO) models had close <i>R</i>² values with the PSO’s being greater in general. Maximum adsorption capacity (<i>q</i>_e) was 41.161&#xa0;mg&#xa0;g⁻¹. Although the intraparticle diffusion (IPD) model fit the data well, the <i>C</i> value suggested the involvement of additional mechanisms. Isotherm analysis demonstrated that the Freundlich model provided the better fit (<i>R</i>² = 0.940), with adsorption constants <i>K</i>_F = 10.755 and <i>n</i> = 1.788, indicating a heterogeneous surface and some form of physisorption. These results suggest that unmodified Djulis SMS biochar is a promising candidate for MB adsorption and can potentially be enhanced for more effective wastewater treatment applications.</p> Graphical Abstract <p></p>