Utilization of biomass for production of second generation bioethanol was considered as a way to reduce burdens of fossil fuel in Pakistan. The materials wheat straw, rice straw, cotton stalk, corn stover, and peel wastes were used in this experiment. Various parameters, such as acidic and alkali pretreatment, enzymatic hydrolysis by cellulases, and effect of proteases inhibitors on ethanol production, were examined. Fermentation was completed by the yeasts Saccharomyces cerevisiae and Clostridium thermocellum separately, and their ethanol production were compared and maximum ethanol yield was obtained with wheat straw i.e.,11.3 g/L by S. cerevisiae and 8.5 g/L by C. thermocellum. Results indicated that a higher quantity of sugar was obtained from wheat straw (19.6 ± 1.6 g/L) followed by rice straw (17.6 ± 0.6 g/L) and corn stover (16.1 ± 0.9 g/L) compared to the other evaluated biomass samples. A higher yield of ethanol (11.3 g/L) was observed when a glucose concentration of 21.7 g/L was used, for which yeast fermentation efficiency was 92%. Results also revealed the increased in ethanol production (93%) by using celluases in combination with recombinant Serine protease inhibitors from C. thermocellum. It is expected that the use of recombinant serpins with cellulases will play a major role in the biofuel production by using agricultural biomass. This will also help in the economics of the biofuel.