Production of bioethanol from oil palm empty fruit bunches (EFB) by Saccharomyces cerevisae and Aspergillus niger is among the ways of reducing environmental pollution and consumption of crude oil. This study used sequential optimization approach based on statistical experimental design including Plackett-Burman (PB) design, one-factor-at-a-time (OFAT) and face-centered central composite design (FCCCD). Among the parameters tested, pH, temperature, inoculum size, potassium dihydrogen phosphate (KH₂PO₄), magnesium sulphate heptahydrate (MgSO₄.7H₂O) and peptone showed positive effects while yeast extract, malt extract, potassium chloride, urea and agitation were influencing the production negatively. The three parameters chosen for determination of the optimum values by response surface methodology (RSM) based on the FCCCD were pH, KH₂PO₄ and agitation. Although agitation showed negative effects it was considered in FCCCD due to the mixing effect of fermnetation. The validity of the model was verified and the optimum value of pH 5.5, agitation of 150rpm and 0.3% of KH₂PO₄ led to a maximum bioethanol production of 7.4 g/l. The yield of bioethanol was determined based on the reducing sugar (16.85 g/l) obtained from the EFB.