Arsenopyrite (FeAsS) oxidative dissolution provides an important source for the occurrence of high arsenic in acid mine drainage (AMD). Biochar is a potent material that can dramatically sequestrate an array of heavy metals in water. However, little is known about the role of biochar on the fate of As from arsenopyrite in AMD. This study investigates the effects of biochar concentrations, AMD acidities, and temperatures on the release of As from arsenopyrite in a simulated AMD over a range of environmentally relevant conditions. Results show that biochar inhibits As release and further acidification without changing the arsenopyrite weathering mechanism. Arsenopyrite is first oxidized to Fe(II), As(III) and S0 and ultimately oxidized to Fe(III), As(V) and SO4 2-, respectively. Higher concentration, temperature or higher acidity promotes the arsenic release rate. Electro-chemical studies showed that biochar inhibited As release and acidification for reduced the charge transfer resistance at the double layer and film resistance at the passivation layer, which was mainly attributed to Fe(III) ions in AMD being adsorbed, oxidized, and As complexed to biochar-Fe-As(V). This study reveals the release mechanism of As from arsenopyrite weathering in AMD and suggests the applicability of biochar in mitigating arsenic pollution and further acidification in sulfide mineral mine drainage.