In order to get some efficient microbial membrane, a Bacillus subtilis (BA1304) was isolated from wastewater, and was identified by the MicroStation System, and was used as the sensitive material for the BOD sensor (BODs) after being immobilized. Three immobilized mediators: polyethylene, sodiumalginate and polyacrylamidewere tested for choosing proper cell carriers. Furthermore, four temperature grads (25ï°C, 30ï°C, 35ï°C and 37ï°C) were studied to find the optimal working conditions. The result showed that the polyethylene was proven to be the most efficientmediator to the electrode in the presence of excess glucose/glutamic acid (GGA). The effective culture temperature was 35ï°C for Bacillus subtilis by testing values in standard BOD solutions, which were estimated using the BODs with deviation less than 5%. The BOD values were determined using the sensor and compared favorablywith those determined by the conventional BOD5method.