Herein, we present the synthesis of surface-oxidized cellulose nanofiber (CNF) hydrogel and characterization with various physicochemical analyses and spectroscopic tools as well as its suitability for cellular encapsulation and delivery. The structure-property relationship as shear thinning, thixotropy, creep-recovery and stimuli responsiveness are explored. The CNF hydrogel is capable to inject possessing shear thinning behavior at shear rate (~10 s−1) range in the normal injecting process. In time-dependent thixotropy, the hydrogel showed rapid transform from flowable fluid back to structured hydrogel fully recovering in less than 60 s. The presence of cell-culture media did not alter shear thinning behavior of CNF hydrogel and showed increased thixotropicity with respect to the control gel. The CNF hydrogel forms 3D structures, without any crosslinker, with a wide range of tunable moduli (~36–1000 Pa) based on concentration and external stimuli. The biological characteristics of the thixotropic gels are studied for human breast cancer cells and mouse embryonic stem cells and indicated high cell viability, long-term survival, and spherical morphology.