This study aimed to investigate the effect of adding CO2 as an impregnation agent in steam explosion on oil palm empty fruit bunch (EFB) for bioethanol production. The influence of this treatment on the characteristics of EFB, enzymatic hydrolysis, and fermentation of EFB was evaluated in this investigation. CO2-added steam explosion was conducted varying the CO2 impregnation time (0, 30, 60 min). The results showed that the addition of CO2 in steam explosion increased the surface area, pore area, and pore volume of EFB. Furthermore, this treatment enabled obtaining yields of glucose and ethanol of 84.14% and 56.01%, respectively, for 60 min CO2 impregnation time. These results were higher than the glucose and ethanol yields of the sample treated by conventional steam explosion, which reached 58.12% and 41.37%, respectively. The findings illustrate the possibility of applying CO2-added steam explosion (CO2SE) for increasing the efficiency of biomass conversion.