In the present study, supercritical carbon dioxide (scCO2) was utilized as a waterless pulping for the isolation of cellulose nanocrystals (CNCs) from waste cotton cloths (WCCs). The isolation of CNCs from the scCO2-treated WCCs’ fiber was carried out using sulphuric acid hydrolysis. The morphological and physicochemical properties analyses showed that the CNCs isolated from the WCCs had a rod-like structure, porous surface, were crystalline, and had a length of 100.03 ± 1.15 nm and a width of 7.92 ± 0.53 nm. Moreover, CNCs isolated from WCCs had a large specific surface area and a negative surface area with uniform nano-size particles. The CNCs isolated from WCCs were utilized as an adsorbent for the hexavalent chromium [Cr(VI)] removal from aqueous solution with varying parameters, such as treatment time, adsorbent doses, pH, and temperature. It was found that the CNCs isolated from the WCCs were a bio-sorbent for the Cr(VI) removal. The maximum Cr(VI) removal was determined to be 96.97% at pH 2, 1.5 g/L of adsorbent doses, the temperature of 60 °C, and the treatment time of 30 min. The adsorption behavior of CNCs for Cr(VI) removal was determined using isothermal, kinetics, and thermodynamics properties analyses. The findings of the present study revealed that CNCs isolated from the WCCs could be utilized as a bio-sorbent for Cr(VI) removal.