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Pretreatment of lignocellulosic biomass by breaking down its complex structure is an essential step to facilitate enzymatic hydrolysis of lignocellulose and subsequent bioethanol fermentation. Herein, an eco-friendly pretreatment strategy was developed by applying deep eutectic solvent (DES) cocktail. Attributing to the synergistic effect of two DESs (e.g., betaine-glycerol and choline chloride-oxalic acid (or choline chloride-acetamide)), an impressive 89.51% digestibility for glucan and 75.43% for xylan were achieved. Characterization of pretreated corn stover showed that DES cocktail changed substrate surface structure and enlarged structure pores, which could improve the accessibility of cellulase. The pretreated corn stover was also employed in ethanol fermentation and achieved the yield of 73.35%. Recycling experiments demonstrated the potential reusability of DES cocktail in a sustainable manner towards various biomass sources. These findings highlight the effectiveness and versatility of the developed DES cocktail pretreatment method, paving the way for sustainable bioethanol production from lignocellulosic feedstocks. [Display omitted] • Betaine-glycerol based DES cocktail was developed for corn stover pretreatment. • DES cocktail pretreatment increased glucan recovery up to 13.1% compared to single DES pretreatment. • The saccharification rate of enzyme-digested glucose after optimization of pretreatment temperature was as high as 89.51%. • Ethanol yield reached 73.61% with a maximum increase of 10.16% after DES cocktail pretreatment. • DES cocktails maintained ∼83% pretreatment effect after four recovery cycles. [ABSTRACT FROM AUTHOR]