The bio hydrogen (H2 ) production by anaerobic digestion of industrial waste is beneficial one due to the availability of proteins and carbohydrates as potential substrate for biological H2 production. An anaerobic fermentative route is a promising method of biohydrogenation. The microbial isolates from various industrial wastes (dairy, sugar and food) were assessed for their potential bio H2 production. The selected individual isolates (F1 - Bacillus subtilis and A3 -Bacillus subtilis) and their cocultures were used for the optimization of bio H2 production utilizing various concentration of biscuit industry waste as substrate at various pH conditions. The mixed consortium which displayed the higher bio H2 production was selected for the detailed analysis of the 3L fermentation studies using 90% Organic Loading Rate (OLR) substrate at pH 6.5. Significantly higher Hydrogen Yield (HY) of 0.87 mol H2 /mol glucose on 16th day of incubation was observed.