This study investigated an innovative strategy of incorporating surfactants into alkaline-catalyzed glycerol pretreatment and enzymatic hydrolysis to improve lignocellulosic biomass (LCB) conversion efficiency. Results revealed that adding 40 mg/g PEG 4000 to the pretreatment at 195 °C obtained the highest glucose yield (84.6%). This yield was comparable to that achieved without surfactants at a higher temperature (240 °C), indicating a reduction of 18.8% in the required heat input. Subsequently, Triton X-100 addition during enzymatic hydrolysis of PEG 4000-assisted pretreated substrate increased glucose yields to 92.1% at 6 FPU/g enzyme loading. High-solid fed-batch semi-simultaneous saccharification and co-fermentation using this dual surfactant strategy gave 56.4 g/L ethanol and a positive net energy gain of 1.4 MJ/kg. Significantly, dual assistance with surfactants rendered 56.3% enzyme cost savings compared to controls without surfactants. Therefore, the proposed surfactant dual-assisted promising approach opens the gateway to economically viable enzyme-mediated LCB biorefinery.