Biofuel is known to be a cleaner and sustainable fuel compared to fossil fuels making it more attractive to be used as transportation fuel. Isobutanol, a four-carbon alcohol has a range of physical properties that are more suitable to be used as gasoline substitute than bioethanol. Isobutanol production by Saccharomyces cerevisiae is reported in small amount. In order to improve the production of isobutanol, experiments on the optimization of media compositions were conducted in this study. Seven critical nutrients affecting isobutanol production were screened using fractional factorial design. The nutrients involved were glucose (C6H12O6), ammonium sulfate ((NH4)2SO4), yeast extract, peptone, potassium phosphate (KH2PO4), magnesium sulphate heptahydrate (MgSO4.7H2O) and iron sulphate heptahydrate (FeSO4.7H2O). The screening process shows that glucose, peptone and yeast extract contributed significantly in enhancing the isobutanol yield. The significant factors were further optimized using Central Composite Design (CCD) and Response Surface Methodology (RSM) with the range of glucose (80-140 g/L), peptone (4-8 g/L) and yeast extract (4-8 g/L). From the experimental results,173 mg/L of isobutanol concentration was obtained at the optimum medium compositions of glucose (140 g/L), peptone (8 g/L) and yeast extract (8 g/L). Throughout the study, it can be concluded that isobutanol yield can be maximize via optimization of medium fermentation. With the improvement of technologies nowadays, the isobutanol production is expected to be increased in the future, encouraging the usage of this fuel in the transportation sector worldwide.