Cellulose nanofibrils (CNF), as a kind of renewable, sustainable and biodegradable natural-based nanomaterial, have shown great potential application in numerous fields due to their fascinating properties. Some properties of CNF-based materials are closely related to their cellulose polymorph and crystallinity index. In this work, we present a facile method for the generation of CNF with tunable polymorphs and crystallinity via the alkali treatment of jute fibers under various conditions followed by (TEMPO)-mediated oxidation and mechanical disintegration. The changes of the cellulose polymorphs and crystallinity induced by alkali treatment on the jute fibers and consequently obtained CNF were well investigated by synchrotron radiation wide-angle X-ray scattering (SR-WAXS), Fourier transform infrared spectra (FTIR) and differential scanning calorimetry (DSC), respectively. Moreover, the morphology of the as-prepared CNF was also examined by transmission electron microscopy (TEM). It has been found that the CNF showed an identical cellulose polymorph to their source material, but a much higher crystallinity index, which revealed the feasibility of CNF generated from jute fibers with tunable polymorphs and crystallinity indexes.