Aiming at solving the problem of the water pollution by acid waste water discharged directly from the coal mine, this study enriched sulfate-reducing bacteria (SRB) from river sludge as a screening sample, separated and purified SRB using dilution coating-lamination plate anaerobic culture method, and studied the removal effects of Fe2+ and SO42- in acid mine drainage from coal mine by SRB. The results showed that the SRB obtained from the isolate coincided with the typical “S” growth curve. The optimum growth conditions of SRB was 34 ℃, pH was seven, and 70% sodium lactate was added as carbon source. The treatment effect on acid mine drainage from coal mine of 10% SRB was optimum by comprehensive comparison of SRB different inoculation amount. The removal rates of SO42- and Fe2+ in coal mine wastewater were 74.71% and 99.18%, respectively, when the inoculation time was five days.