The aim of this work was to study the physico-chemical pretreatment and enzymatic hydrolysis of cotton stalk for ethanol production by Saccharomyces cerevisiae. Firstly, factors affecting pretreatment were screened out by Plackett-Burman design (PBD) and most significant factors were further optimized by Box-Behnken design (BBD). As shown by experimental study, most significant factors were FeCl3 concentration (FC), irradiation time (IT) and substrate concentration (SC) affecting pretreatment of cotton stalk among all studied factors. Under optimum conditions of pretreatment FC 0.15 mol/l, IT 20 min and SC 55 g/l, the release of reducing sugar was 6.6 g/l. Hydrolysis of pretreated cotton stalk was done by crude on-site produced enzymes and hydrolysate was concentrated. Ethanol production by Saccharomyces cerevisiae using concentrated cotton stalk hydrolysate was 9.8 g(p)/l, with ethanol yield 0.37 g(p)/g(s) on consumed sugars. The data indicated that microwave FeCl3 pretreated cotton stalk hydrolyses by crude unprocessed enzyme cocktail was good, and ethanol can be produced by fermentation of hydrolysate.