Highly carboxylated nanocellulose fibers can be functionalized with cell adhesive peptides and cationic cross-linked to form matrices for a three-dimensional (3D) cell culture. It is hypothesized that nanocellulose hydrogels cross-linked with divalent cations can provide the required biochemical and mechanical properties for intestinal organoid growth and recovery. Nanocellulose hydrogels are produced by TEMPO- and TEMPO-periodate-mediated oxidation and functionalized with RGD peptides. Mechanical properties are measured by rheology and optical properties quantified by UV-vis spectroscopy. Cellulosic matrices are cross-linked with Ca2+ and Mg2+ and intestinal organoids cultured for 4 days. The organoids are recovered for passaging and RNA extraction. TEMPO-periodate-oxidized nanocellulose fibers form functionalized hydrogels and support the growth of intestinal organoids. The highly transparent cellulosic matrix requires 4 times more Mg2+ than Ca2+ ions to reach the targeted stiffness. Organoids cultured in nanocellulose maintained a major living area for up to 4 days. Cell clusters recovered from magnesium-cross-linked hydrogels can be passaged, and their extracted RNA is intact. Cationic cross-linked nanocellulose hydrogels are promising alternative plant-based matrices for a 3D cell culture systems.