The present work emphasizes the isolation of cellulose nanofiber (CNF) from the kenaf (Hibiscus cannabinus) bast through a chemo-mechanical process. In order to develop high CNF yield with superior properties of CNF for improving compatibility in varied applications this method is proposed. The fiber purification involved pulping and bleaching treatments, whereas mechanical treatment was performed by grinding and high-pressure treatments. The kraft pulping as a delignification method followed by bleaching has successfully removed almost 99% lignin in the fiber with high pulp yield and delignification selectivity. The morphology of the fibers was characterized by scanning electron microscopy, which showed a smooth surface, fiber bundles, gel-shaped nanofiber, and an average size of 94.05 nm with 69% of CNF in 34-100 nm size. The chemo-mechanical process exhibited a more crystalline nature in CNF than pulp kenaf. The low zeta potential values exhibit the distribution of fibrils and colloidal suspension stability without any further agglomeration. A lower concentration of CNF is less stable exhibiting the product agglomeration. Therefore, the chemo-mechanical process for the isolation of CNF (Hibiscus cannabinus) from kenaf involves sustainable, low-cost, non-toxic, and cheap alternatives than other traditional methods.