The objective of this study is to elucidate sorption mechanism of ammonia-N by Spent Mushroom Substrate Biochar (SMSB), produced from pyrolysis process of Spent Mushroom Substrate (SMS) at 550°C for two hours. Surface functional group, ash contents, and elemental compositions were analysed using Boehm titration, furnace and Inductively Coupled Plasma-Optical Emission Spectroscopy (ICPOES) respectively. Sorption parameters (i.e., sorbent dosage, input ammonia-N concentration, and contact time) were optimised, and the sorption data were analysed using kinetic models (i.e., pseudo-first-order and pseudo-second-order), and isotherm models (i.e., Langmuir, Freundlich, and Dubinin-Radushkevich (D-R)). The surface of SMSB consists of carboxylic (0.3 mmol/g), phenolic (0.5 mmol/g), and basic functional groups (0.2 mmol/g). Sorption of ammonia-N was best described by the pseudo-second-order kinetic model and the Dubinin-Radushkevich (D-R) isotherm model. The D-R sorption capacity for SMSB was 12.6 mg/g, and correlates well with the amount of acid functional groups (i.e., carboxylic and phenolic groups) in SMSB (i.e., 14.4 mg/g). The low Freundlich constant (n = 0.754), and low D-R mean free energy per mol of the adsorbate (E = 4.4 kJ/mol) indicates a physical sorption process, and ammonia-N binds with acid functional groups through weak forces. The result shows great potential of SMSB for removing ammonia-N in wastewater.