Abstract: To explore the effects of COD_Cr/SO₄^2- and substrate on the removal of sulfate, the difference of the sulfate removal to five different substrates, i.e. lactic, ethanol, propionic, butyric and acetic, under different COD_Cr/SO₄^2- ratios was studied in the conical flask with static condition. Results reveal that SRB with the substrate of ethanol has higher affinity than any other substrates at the COD_Cr/SO₄^2- of 2.0 at the temperature of 35 ℃. When the COD_Cr/SO₄^2- increases to 2.5, the system with lactic, ethanol and propionate as substrate rapidly reaches high sulfate removal rate, being 90.29%, 81.27% and 80.69%, respectively. At the COD_Cr/SO₄^2- of 3.0, the system with butyrate and acetate as substrate has high sulfate-reducing rate, being 88.78% and 73.69%, respectively. It is shown that COD_Cr/SO₄^2- rate has significant effects on the consumption of different substrates in the anaerobic system. Therefore, keeping the system at optimal COD_Cr/SO₄^2- according to the substrates is an effective approach to increase the sulfate-reducing rate. This also provides a new thought to shorten the set-up time of anaerobic sulfate-reducing reactor.