Lignocellulosic substances such as agricultural wastes are attractive feed stocks for bioethanol production. Indica IR.64 rice straw is one of abundant agricultural wastes in Indonesia and could be used to bioethanol production. It has several characteristics such as high content of cellulose and hemicelluloses that can be readily hydrolyzed into fermentable sugars. A simple process (the direct saccharification and fermentation process) to produce ethanol from rice straw was developed in order to establish an efficient bioethanol production. In this work, no harsh pre-treatment steps were applied and also use a simple one-vat reactor without the risk of losing liberated carbohydrate. The first step in using rice straw for bioethanol production is size reduction through milling and sieving process prior to enzymatic hydrolysis. Direct saccharification and fermentation (DSF) of Indica IR.64 rice straw was examined and compared with two type of control (systems devoid of yeast and enzyme). The experiment were carried out under anaerobic condition, where the cellulase crude enzyme and cellulosic substrates (rice straw) produced glucose from the cellulose and Saccharomyces cerevisiae directly assimilated the glucose to bioethanol. The faster rate of bioethanol production during DSF by Saccharomyces cerevisiae was obtained within the first 12h. The maximum ethanol concentration, ethanol yield, and theoretical ethanol yield of untreated rice straw were 0.25 g/L, 10 and 14.88%, respectively. Nevertheless, the direct saccharification and fermentation shows the potential for lower cost and higher efficiency for bioethanol production.