The present study aims at investigating the application of ultrasound assisted choline chloride (ChCl) - formic acid (FA) deep eutectic solvent (DES) pretreatment of Barley straw. In addition, the efficiency of a wet grinding followed by high intensity ultrasound (HIUS) treatment for production of cellulose nanofibers (CNF) has been evaluated. The DES (using ChCl: FA at 1:9 M ratio) treatment at 45 kHz ultrasound frequency and 3 h of treatment duration resulted in 84.68 ± 1.02 % and 82.96 ± 0.79 % of lignin and hemicellulose solubilisation, respectively. The purification of DES treated solid residue resulted in cellulose with more than 90 % purity. Further, 10 min of wet grinding followed by 40 min of HIUS treatment resulted in more than 80 % nano-fibrillation efficiency. The produced CNF had diameters less than 100 nm in number size distribution and type I cellulose structure. This study confirmed that the developed process offers a sustainable method for producing nanocellulose from agricultural waste.