Hypothesis: Cellulose nanofibres produced by TEMPO-mediated oxidation can form gels. This study presents a proof-of-concept for gel electrophoresis with nanocellulose (NC). Experiments: TEMPO-oxidised cellulose nanofibre dispersion is chemically cross-linked by inducing amide linkages to produce gel slabs for electrophoretic separation. Nanocellulose gel slabs 1 cm thick containing Tris/Borate/EDTA (TBE) buffer were casted. Different cross-linker types and ratios are investigated to assess the migration of conventional electrophoresis tracking dyes. Findings: Tracking dyes (bromophenol blue and orange G) can diffuse within the gel at different rates and therefore separate. Changing the cross-linker length from EDA to HMDA (C2- to C6-chain) increases the overall network pore size resulting in a faster migration rate for both bromophenol blue and orange G. Increasing the cross-linker concentration stabilises the HMDA-NC gel (no extension) during the electrophoresis run without any effect on the dye migration rate. Increasing the voltage increases the migration rates for both orange G and bromophenol blue. Further development is required to cast the gels evenly and to prevent bubble formation during the cross-linking process. This will enable to effectively separate mixtures of proteins. Nanocellulose gels can become a novel substrate for sustainable biomedical separation and diagnostics by electrophoresis. Crown Copyright (C) 2019 Published by Elsevier Inc. All rights reserved.