Abstract: As a major by-product of Zanthoxylum bungeanum, pepper seeds are rich in cellulose and serve as an excellent biomass material. In recent years, Zanthoxylum bungeanum seeds have been in a state of low-value utilization, often discarded as agricultural waste, and the rich cellulose resources have not been rationally utilized. Alkali dissolution method, as a conventional method for preparing cellulose, has shortcomings of difficult recovery of the chemical reagents and large environmental pollution, although it has a high yield and a wide range of applications. At the same time, eutectic solvents, as a new type of solvent, have the advantages of green environmental protection, designable structure, good extraction effect and low price, and have been widely used in the separation and extraction of plant components. To fully exploit their utilization potential, this study introduced an innovative approach combining ultrafine grinding pretreatment with deep eutectic solvent (DES) for the preparation of cellulose from Zanthoxylum seeds. The effects of key parameters, including ultrafine grinding time, DES system composition, and solid-to-liquid ratio were systematically investigated using cellulose content and hemicellulose/lignin removal rates as the evaluation indices. The optimal preparation process was subsequently established. Furthermore, the structural characteristics of the obtained cellulose were analyzed using X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and Scanning electron microscope (SEM). The results demonstrated that the optimal conditions for preparing pepper seed cellulose by ultrafine grinding-assisted DES were as follows: choline chloride-oxalic acid (ChCl-OA) molar ratio of 1:2.2, solid-to-liquid ratio of 1:17 g/mL, and processing time of 5.24 hours. Under the optimal conditions, the cellulose content reached 89.24%, significantly higher than that achieved by the conventional alkaline method with the content of 66.58%. The cellulose obtained through the ultrafine grinding-assisted DES method displayed key performance advantages compared to cellulose prepared by conventional alkaline dissolution. Compared with the cellulose prepared by the conventional alkali solution method, the particle size of cellulose prepared by eutectic solvent was 16.30 μm, and there was no significant difference in particle size between the two cellulose (P> 0.05). In terms of macroscopic color, cellulose prepared by eutectic solvent was more black, green and blue, and had higher water and oil holding capacity of 7.63 g/g and 4.37 g/g, respectively. From the XRD and thermogravimetric results, cellulose prepared by eutectic solvent has a high degree of polymerization (62.61% crystallinity) and good thermal stability. According to the FTIR results, the characteristic absorption peaks of cellulose prepared by eutectic solvent at 779 cm⁻¹ and 1738 cm⁻¹ indicated the formation of cellulose oxalate. In terms of micromorphology, both cellulose forms microcrystalline aggregates to a certain extent. This study not only establishes an efficient and eco-friendly approach for valorizing pepper seed by-products but also provides insights into the mechanism of DES-mediated lignocellulose fractionation. The combination of ultrafine grinding and DES offers a sustainable alternative to traditional chemical methods, minimizing energy consumption and avoiding toxic reagents. The high-purity cellulose obtained holds potential for applications in biodegradable materials, food additives, and pharmaceutical excipients, thereby contributing to the circular economy and resource efficiency in agricultural processing. Further research could explore the scalability of this process and its adaptability to other lignocellulosic biomass sources.