The substrate of rice (Oryza sativa) straw employed in this study was procured from local market and stored in polyethene bags until needed. The study was conducted to find out the effects of process optimization of ethanol production from enzymatically saccharified biomass of acid-pretreated rice straw. The results indicated that acid pretreatment has reduced lignin content about 50%. Resultantly, pretreatment raised cellulose contents up to 52% making it suitable for enzymatic saccharification. Optimization of saccharification illustrated that parameters such as biomass, enzyme concentration, temperature, incubation time and agitation indeed affected the action of cellulase on pretreated biomass releasing sugars up to 31.0g/L. The results clearly indicate the need for optimization at every point in the ethanol production process for efficient use of resources and maximum yield without wastage. Among various operational modes of fermentation studied, such as batch, semi-continuous and continuous, the semi-continuous process gave better ethanol yield of 23.4 g/L. This implies potential use of the process in industrial grade ethanol production. Collectively the study has shown that rice straw can serve as a lignocellulosic substrate, utilizing acid pretreatment for cellulose exposure and enzymatic saccharification to release fermentable sugars for production of ethanol.