There are very few reports, if any, on the physicochemical changes during the powder fabrication of cotton gin trash (CGT), an abundant source of lignocellulose. This particular study can be very informative for the production of new CGT composite materials. In our current study, we investigated the changes in particle size, morphology, surface area, chemical structure, crystallinity, thermal and moisture properties of CGT at different stages of the milling operation. An attritor milling of CGT up to 4 h reduced the particle size (from 144.2 μm to 6.2 μm) and enhanced the surface area up to 2.5 times without damaging its original properties, including crystallinity and chemical structure. Milling operation beyond 4 h did not further reduce the particle size effectively, though affected the thermal stability of CGT. Overall, it is suggested that the milled CGT can be a sustainable source of lignocellulose powder for use in a wide area of applications.