The extracellular matrix (ECM) plays a critical role in tumor progression by regulating cancer cell behavior, invasion and metastasis. This importance has motivated the development of biomaterials that closely mimic the native tumor ECM to create more accurate and predictive in vitro cancer models. In this study, oxidized cellulose nanofibrils (OCNF) are used as a simple three-dimensional scaffold for breast tumor spheroid expansion. OCNF forms a highly dynamic hydrogel in cell culture medium with rheological properties suitable for modeling early breast tumor ECM. This dynamic environment enables MCF-7 cells to form spheroids within 6 days and to continue proliferating for up to 12 days. Co-culture of MCF-7 cells with fibroblasts further models the tumor microenvironment with cancer-associated fibroblasts (CAFs). Because the hydrogel is held together by non-covalent interactions, adding extra cell culture medium and pipetting converts it into a fluid-like state, facilitating spheroid release and collection for downstream applications such as drug screening and penetration studies. Overall, this work demonstrates a facile approach for preparing tumor spheroids using cellulose nanofibril hydrogels and may inspire further innovations in biomedical applications of cellulose hydrogels.