ABSTRACT: Sugarcane bagasse (SCB) is an abundant agricultural waste in China and the conversion of the waste into plethora of useful resources is very vital. To achieve this, fractionation of the waste is highly important in the biomass biorefinery. The present study aims at investigating the synergistic role of deep eutectic solvents (DES) with sweeping frequency ultrasound (SFUS) and fixed frequency ultrasound (FFUS) in the fractionation of SCB to enhance the enzymatic saccharification process. Therefore, the effects of ultrasound (US) and DES conditions on the pretreatment efficiency were investigated. Under optimum SCB pretreatment conditions, FFUS (40 kHz, 60 min) + DES (choline chloride (ChCl)-lactic acid (LA), 120 °C, 3 h) and SFUS (40 kHz, 60 min) + DES (ChCl-LA, 120 °C, 3 h), the lignin removal rates were 80.13 and 85.62%, respectively. The hemicellulose removal rates were 78.08 and 90.46%, respectively; and the contents of glucose, xylose and arabinose in the liquid fractions after FFUS + DES pretreatment were 7.07, 17.95 and 3.01%, respectively. However, the yield of glucose, xylose, and cellobiose after enzymatic hydrolysis of the SFUS + DES pretreated SCB were 86.76, 38.68, and 20.76%. Analytical studies revealed that the SFUS + DES pretreatment can effectively destroy the ultrastructure of SCB and reduce the crystallinity of cellulose. Furthermore, the mechanism of pretreatment with SFUS + DES was proposed, which confirmed the excellent performance of SFUS + DES. Thus, the application of SFUS + DES pretreatment was able to improve the removal of lignin and hemicellulose from SCBs.