Biochars have demonstrated great potential for water decontamination and soil remediation; however, their redox reactivity toward trace contaminants and the corresponding redox-active moieties (RAMs, i.e., phenolic −OH, semiquinone-type persistent free radicals (PFRs), and quinoid CO) remain poorly understood. Here we investigated the roles of the RAMs on biochar in oxidation of As­(III) under varying pH and O2 conditions. The results showed that the promoted oxidation of As­(III) by the RAMs is strongly pH dependent. Under acidic and neutral conditions, only the oxidation of As­(III) by •OH and H2O2 produced from activation of O2 by phenolic −OH and semiquinone-type PFRs occurred. In contrast, the oxidation by semiquinone-type PFRs, quinoid CO, and H2O2 (if O2 was introduced) appeared under alkaline conditions. This pH-dependent oxidation behavior was attributed to the varying redox activities of RAMs, as confirmed by multiple characterization and validation experiments using biochar with tuned RAMs compositions, as well as thermodynamics evaluation. Our findings provide new insights into the roles of the RAMs on biochar in the promoted oxidation of trace As­(III) over a broader pH range under both anoxic and oxic conditions. This study also paves a promising way to oxidize As­(III) with biochar.