In this study, the biochar (BC)-induced Fe(III) reduction for persulfate (PS) activation system was used for the first time in sulfamethoxazole (SMX) degradation. The apparent rate constant of the BC/Fe(III)/PS system was 31.3 and 8.2 times that of BC/PS system and Fe(III)/PS system, respectively. The carbon-centered persistent free radicals contained in BC acted as electron donors. Electron transfer occurred between the BC surface and Fe(III) successfully. Fe(II) was thus generated and played a key role for the subsequent PS activation. Scavenger quenching experiments and electron spin resonance spectrometry confirmed the presence of sulfate (SO4-) and hydroxyl radicals (HO center dot), which brought about the efficient SMX degradation in the BC/Fe(III)/PS system. Furthermore, the frontier molecular orbital (FMO) theory and dual descriptor (DD) method were employed in predicting radical attacking sites of SMX. According to the results of theoretical computations and the experimental detection, degradation pathways of SMX in the BC/Fe(III)/PS system were deduced accurately. This study gives new insight into the possible mediatory roles of biochar in PS activation by transition metal ions. The BC/Fe(III)/PS system is a promising process for groundwater and soil remediation.