In the present study E .coli was selected for the fermentative hydrogen production. The E. coli strain was isolated from sewage sample and identified based on gram staining and growth on selective media (EMB agar). Isolated E. coli was used for hydrogen production using sweet sorghum juice and sugarcane juice. The hydrogen produced by E. coli was estimated by water displacement method. The more amount of water was being displaced (420 ml) in 24 h by E. coli using sugarcane juice. Whereas 390 ml of water was displaced where the sweet sorghum juice used as a substrate. More amount of water was displaced (420 ml) by E. coli using sugarcane juice at the pH of 6.5 when compared with pH 4.5 (315 ml), pH 5.5(390ml),pH 6.5 (420 ml), pH 7.5 (400 ml) and atpH 8.5 (320 ml). The result revealed that the E. coli can produce more amount of hydrogen using sugarcane juice as substrate at the pH range from 5.5 (390 ml) to 6.5(420 ml).