Objective To investigate the function of a novel biosensor used for the rapid determination of biochemical oxygen demand (BOD) which is developed by our research group based on suspended immobilized microbial cell system in a completely mixed determining chamber as a substitute of the traditional membrane system. Methods Activated sludge was immobilized by PVA gel and used as a bio-sensing element. The novel biosensor was used to measure the short time BOD value and the conventional cultivation method was used for BOD5 measurement. Results A linear relationship was observed for the difference between the current and the concentration of glucose-glutamic acid (GGA) solution below 200 mg/L with a correlation coefficient of 0.995. The optimal response of the sensor was obtained at pH 7.0 and 30℃. The sensor response was within 15 min and was reproducible within ±5% of the mean in a series of eight samples containing 75 mg/L BOD using standard GGA solution. The novel sensor response was found to be fairly constant over a period of 30 days, with ±5% fluctuations. Conclusion A relatively good agreement is found between BOD estimated by the novel BOD biosensor and that determined by the conventional 5-day BOD method. This novel BOD biosensor has good sensitivity, stability and reproducibility.