Hydrogen production by immobilized Enterobacter aerogenes on the carbon fiber (CF), surface modified carbon fiber, granular and powdered activated carbon in batch mode of operation was investigated. The surface morphology and chemical properties of CFs were characterized by field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). Among the supports employed, immobilization on treated CF (CF-T) resulted in increase of both hydrogen production rate (HPR) and hydrogen yield (HY). The present study showed that immobilized E. aerogenes on 0.2 mg/mL CF-T resulted in HY of 2.56 mol/mol glucose and HPR of 2.48 L/L.h representing 23%, and 34%, enhancement compared to the free E. aerogenes, respectively. Using powdered activated carbon as immobilization support resulted in HY of 2.13 mol/mol glucose, which was higher in comparison to HY of granular activated carbon by 1.33 mol/mol glucose.