The present work studied the effects of alkali pretreatment on the cellulosic biomass of rice straw. The improvement in the cellulose content and reduction in the lignin and hemicellulose percentage was observed with alkali pretreatment. Fourier transformation infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM) analysis confirm the modification in the surface structure of alkali rice straw. Further, the study investigated the potential of different types of seed sludge as inoculum sources for dark fermentative biohydrogen production. In comparison to other sludge samples (beverage industry, food industry, and sewage treatment plant sludge), the mixed culture of sewage treatment plant sludge had the highest cumulative volume of biohydrogen (90.52 mL), as well as the highest hydrogen production yield (0.75 moleH2/mole) with the substrate utilization of 86.72%. The results provide information on the best sludge source for enhancing biohydrogen production in the dark fermentation method.