Herein, a natural deep eutectic solvent (DES) is developed to deconstruct the recalcitrant structure of sugarcane bagasse (SCB) for further 5-hydroxymethylfurfural (5-HMF) production. The properties, structure of the DES, and the influence of pretreatment and reaction conditions on SCB are studied. The findings reveal that carboxylic acid DES pretreatment results in a substantial decrease of hemicelluloses and lignin. At the same time, carboxylic acid DES systems significantly improve the yield of 5-HMF at 110 °C. Under the optimum conditions (choline chloride (ChCl)–lactic acid (8.33 wt% H₂O), SCB/DES (1:10/wt:wt), temperature 110 °C, and time 4 h), the 5-HMF yield obtained from SCB is 56.68 mol% (1.62 mg mL⁻¹). This 5-HMF yield is significantly higher by 25.88, 16.43, and 9.87-folds compared to the 5-HMF yield from the SCB treated with ChCl–urea, ChCl–glycerin, and ChCl–ethylene glycol, respectively, under the same reaction conditions. In addition, component analysis and structural characteristics suggest that the content of lignin in the SCB decreases significantly, and the cellulose and hemicellulose are partially hydrolyzed after being treated with the DES composed of ChCl and carboxylic acids. Overall, this work shows that SCB pretreatment with a carboxylic acid DES is promising for low-cost biorefineries to achieve effective biomass fractionation and conversion.