Nanofibrils of cellulose were prepared from short staple cotton by refining process using a lab disc refiner that exerts a combination of shear and frictional forces. The nanofibrils were characterized by scanning electron microscope (SEM), atomic force microscope (AFM), X-ray diffractometer (XRD) and Fourier transform infrared spectroscopy (FTIR). From SEM and AFM, it was found that starting average diameter of the cotton fiber (~25 ${mu}m$) was reduced to 242 nm after 30 passes of refining. FTIR analysis revealed the increase in amorphous nature of cotton cellulose due to refining process. Supportively, XRD analysis showed a steady decline in percent crystallinity of the cotton fibers as the cotton fibres were passed through the refiner for more number of passes. Similarly, degree of polymerization (DP) was reduced from 2720 to 740 due to the refining process. Nanofibrils of cellulose from short staple cotton have a huge potential for application in nanofilters and as biodegradable fillers in nanocomposites.