AbstractDeep eutectic solvent (DES) is a new type of green solvent that can be used for the separation of lignocellulosic components. In this study, ultrasound‐assisted DES pretreatment was used to treat cotton straw, which could retain most of the cellulose in cotton straw and separate hemicellulose and lignin at the same time. Here, DESs with different molar ratios were prepared using choline chloride as hydrogen bond acceptor and formic acid as hydrogen bond donor. Simultaneously changing the ultrasonic frequency and DESs synergistic extraction of lignin. Results show that the ultrasonic power is 32 kHz (room temperature), ultrasound 30 min, oil bath heat 3 h under the best conditions can significantly extract lignin, and the yield of lignin is 87.7% and the purity reached 97.3%. The characteristic peaks of FT‐IR confirmed the extraction of lignin. The structural characteristics of 2D‐HSQC also confirmed the effective separation of lignin, and x‐ray diffraction (XRD) and scanning electron microscope (SEM) confirmed the effect of ultrasound‐assisted DES pretreatment on lignocellulose structure. In short, this study provides a feasible method for the separation of lignocellulose.