The effluent of biogas production plant was used for the pretreatment of rice straw for the improvement of ethanol production. In addition, the organic active ingredients of the effluent, i.e., acetic, butyric, lactic and propionic acids (1-4%), as well as water were employed for the pretreatment at 100 and 140 °C. The results indicated that pretreatment at 100 °C had no significant effect on the performance of subsequent enzymatic hydrolysis and ethanol production by simultaneous saccharification and fermentation (SSF). Among different types of organic acids presented in the effluent, lactic acid showed a better performance. The highest concentration of glucose and ethanol were achieved after 72 h enzymatic hydrolysis and SSF from the straw pretreated at 140 °C with 4% lactic acid. Applying the effluent for the straw pretreatment at 140 °C resulted in an increase in glucose and ethanol concentrations by 42.4 and 47.5%, respectively, compared to those from untreated samples. SEM, FTIR, BET, BJH, and compositional analyses were used to characterize the changes in the structure and composition of rice straw by the pretreatment. Changes in the straw swelling, cellulose crystallinity, pore size distribution, and composition were responsible for the acquired improvements.