This paper investigated hydroxyl radical (OH) generation from biochar suspensions for diethyl phthalate (DEP) degradation in the presence of oxygen. Electron paramagnetic resonance (EPR) coupled with a salicylic acid trapping method were used to detect free radicals in biochar and verify OH generation from biochar suspensions. Free radicals (FRs) in biochar could induce OH generation, and ≈12 spins of FRs were consumed to produce one trapped [OH] molecule. The proposed mechanism of OH generation was that FRs in biochar transferred electrons to O2 to produce the superoxide radical anion and hydrogen peroxide, which reacted further with FRs to produce OH. Free radical-quenching studies utilizing superoxide dismutase, catalase, and deferoxamine as scavengers were used to testify this mechanism. Furthermore, OH generated from biochar suspensions could degrade DEP efficiently. These findings of this study provide new insights into the physicochemical properties and environmental implications of biochar.