Wheat straw is one of the most abundant lignocellulosic feedstocks, contains mixture of sugars, selected for ethanol production. Microwave alkali pretreated wheat straw was used as substrate for ethanol production from Saccharomyces cerevisiae. Optimization of ethanol production from wheat straw enzymatic hydrolyzate was studied using sequential statistical optimization process. First factors affecting ethanol production were screened out by Plackett–Burman design (PBD). Most significant factors pH (4–7), temperature (20–40°C), initial total reducing sugar conc. (TRS) (1–10%) and inoculum level (1–5%) further studied by Box–Behnken design (BBD) for ethanol production. Maximum ethanol production obtained during BBD was 15.6g/L with ethanol productivities 0.43g/L/h. Under optimum conditions ethanol production studied at bioreactor level and obtained ethanol concentration 16.4g/L with ethanol productivities 0.45g/L/h obtained at pH 5.5, temperature 30°C, inoculums level 3.3% and TRS conc. 6.5%. These results indicated that ethanol production can be enhanced by optimization of fermentation conditions.