A high performance, long-term, stable glucose sensor was developed and evaluated for continuous measurement of glucose for use in National Aeronautics and Space Administration space bioreactors while using a rotating wall perfused vessel system to support a baby hamster kidney (BHK-21) cell culture. The sensor employs a glucose oxidase multilayered membrane with a hydrogen peroxide electrode system. The output of the sensor was converted to glucose concentration utilizing the two-point calibration method. The sensor, tested over four 30 day bioreactor cell runs, showed a steady-state response time of less than 15 min. The sensor was able to measure glucose continuously and accurately for 16 days while using a single calibration and 30 days using two sets of calibrations, resulting in a standard error of prediction of +/-0.5 mM as against a benchtop Beckman glucose analyzer. Evaluation conditions like cell culture pH, incubator temperature conditions, and operational flow rate, were found to have a very little effect on the sensor performance.