Nanocellulose (NC) has attracted close attention in research and industry fields because of its excellent properties and plentiful hydroxyl group accessible for surface modification. In this study, the cellulose isolated from peanut shells was used to prepare carboxymethylated nanocellulose (CMNC) under different reaction conditions, and Electron beam irradiation (EBI) pretreatment with different doses (40, 80 and 120 KGy) was previously used before modification to improve the reaction accessibility of nanocellulose. The substitution degree (DS), structural, physicochemical and functional properties of carboxymethyl CMNC with EBI pretreatment were investigated. The results showed that the peanut shell nano cellulose was a typical rod-like structure with a diameter of 5–40 nm and a length of 50–300 nm, the aspect ratio distribution was about 6.37, and EBI pretreatment significantly improved the modification effect of CMNC. The DS of the unirradiated sample was 0.38 and 0.48, and the DS increased to 0.66 and 0.69 at the irradiation dose of 120 kGy, which increased by 74% and 44%, respectively. The carboxymethyl modification changed the morphology of nanocellulose from the original short rod-like to spherical particles, and the effect was more evident with the increase of irradiation dose. FTIR showed that the nanocellulose successfully introduced carboxyl groups, the crystalline structure of nanocellulose was changed by carboxymethyl reaction. The Zeta potential result showed that the dispersion stability of CMNC suspension was improved. These results provide a helpful basis for further investigations on the modification in nanocellulose induced by EBI, verifying the effectiveness and feasibility of using EBI to modify nanofibers. They are expected to guide the transformation of nanocellulose and its most possible applications in the future to improve its performance in different applications.