Biobutanol produced by acetone-biobutanol-ethanol (ABE) fermentation process has been revisited in the light of its use as “drop in” liquid biofuel to be blended with gasoline. In this study, renewable feedstock like rice straw, sugarcane bagasse and microalgal hydrolysate were used in ABE fermentation via separate hydrolysis and fermentation. Clostridium acetobutylicum ATCC 824 was used as the fermenting organism. Alkali pretreatment followed by enzymatic hydrolysis was used for rice straw and sugarcane bagasse. In batch fermentation, a biobutanol titer and yield of 9.10 g/L and 0.42 mol/mol glucose (0.17 g biobutanol/g glucose), respectively was obtained from rice straw, while sugarcane bagasse achieved a biobutanol titer and yield of 8.40 g/L and 0.40 mol/mol glucose (0.16 g biobutanol/g glucose), respectively. Higher microalgal biomass loading with 3% acid pretreatment severely inhibited fermentation performance. Unhydrolyzed microalgal biomass at a loading of 180 g/L in ABE fermentation resulted in 4.32 g/L biobutanol and 0.09 g biobutanol/g microalgae as yield. C. acetobutylicum was immobilized in polyvinyl alcohol (PVA) for improving the cell loading in fermentation and protect the cells from biobutanol toxicity. With rice straw hydrolysate as a feedstock and in the absence of yeast extract, a biobutanol titer, yield and productivity of 13.80 g/L, 0.90 g/L/h, and 0.58 mol biobutanol/mol glucose (0.23 g biobutanol/g glucose), respectively were obtained. Hence, rice straw is a potential feedstock for biobutanol production for fuel use.