To establish an efficient bioethanol production system, a solid-acid catalyst-based saccharification of rice straw in two steps was developed. It was rarely concerned so far that many biomass species contain a significant amount of nonstructural carbohydrates (NSCs, free sugars and starch). NSCs, including hemicellulose, are readily hydrolyzed but also susceptible to overreaction with byproducts. In the developed process, the first step is intended to effectively hydrolyze the significant sugar source of NSCs and HC in rice straw under a mild condition, and the second step is addressed to hydrolyze the cellulose in the residue obtained under a harsh condition. For each step, several catalysts were screened and reaction condition were investigated. For the first step, an appropriate catalyst was Amberlyst 35 Dry, at 130 degrees C for 30 min in which high yields of C6 monosaccharide, 47.2%, and C5 monosaccharide, 10.8%, were obtained. For the second step, the most active catalyst was a sulfonated mesoporous carbon that provided a maximum yield of C6 sugar (52.5%) at 220 degrees C for 0 min. A test with the sequential two-step saccharification provided a 65% yield of sugars. From the investigations, the most studied modified pathways of dilute-acid catalyzed hydrolysis of HC and cellulose were also likely to occur with a solid-acid catalyst with an additional pathway of direct hydrolysis of NSCs to C6. To support the observed phenomena, the characteristics of the RS before and after saccharifications were analyzed using SEM and XRD. Thus, this process represents a method to increase the cost-effectiveness of bioethanol production system in an environmentally sound way.