To design a new system of novel TEMPO-oxidized cellulose nanofibrils (TOCNs)/graphene oxide (GO) composite, 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation was utilized. For the better dispersion of GO into the matrix of nanofibrillated cellulose (NFC), a unique process combining high-intensity homogenization and ultrasonication was adopted with varying degrees of oxidation and GO percent loadings (0.4 to 2.0 wt%). Despite the presence of carboxylate groups and GO, the X-ray diffraction test showed that the crystallinity of the bio-nanocomposite was not altered. In contrast, scanning electron microscopy showed a significant morphological difference in their layers. The thermal stability of the TOCN/GO composite shifted to a lower temperature upon oxidation, and dynamic mechanical analysis signified strong intermolecular interactions with the improvement in Young’s storage modulus and tensile strength. Fourier transform infrared spectroscopy was employed to observe the hydrogen bonds between GO and the cellulosic polymer matrix. The oxygen permeability of the TOCN/GO composite decreased, while the water vapor permeability was not significantly affected by the reinforcement with GO. Still, oxidation enhanced the barrier properties. Ultimately, the newly fabricated TOCN/GO composite through high-intensity homogenization and ultrasonification can be utilized in a wide range of life science applications, such as the biomaterial, food, packaging, and medical industries.