Sulfonamide antibiotic contamination promotes the generation of resistance genes, whereas its removal remains a great challenge due to the lack of understanding in its removal micro-mechanism and efficient adsorbents. In this study, the interfacial interaction mechanism associated with the adsorption of sulfamethazine (SMT) on mesoporous cellulose biochar (MCB) adsorbents were studied, as well as the influences of pH value, cations and humic acid (HA) on this interfacial interaction mechanism. A pi-electron interaction, not electrostatic interactions, is the main interfacial interaction mechanism. A thermodynamic and kinetic study revealed that the monolayer adsorption occurred and was dominated by chemisorption. Density functional theory (DFT) and frontier orbital theory (FOT) simulations were conducted to further explore the interfacial interaction micro-mechanism at the molecular and electronic levels. The adsorption equilibrium configurations confirmed that SMT was adsorbed onto MCB in the V-shape configuration, mainly through pi-pi EDA interactions. DFT simulations showed that increasing the pi electron density of the biochar favored its adsorption of SMT and that the pyrrole groups might be the most effective functionalities for this adsorption. The above findings could provide significant reference value for the design and preparation of efficient sulfonamide antibiotic biochar adsorbents.