Herein, preparation and modification of cellulose nanofibers (CNF) from recycled old corrugated box (OCB) raw material is reported. CNF were synthesized from cellulose microfiber obtained after the pre-treatment of alkaline, acetic acid/sodium chlorite followed by bleaching with hydrogen peroxide. Intermediate fractions during the pre-treatment were collected and examined for the lignin and hemicelluloses contents using FTIR, GPC, GC-MS and HPLC methods. Both the treated and untreated OCB microfibers were analyzed using XRD, TGA, and SEM to confirm the removal of non-cellulosic contents. Resultant pre-treated microfiber was break-down into nanofibers under high-pressure using high-pressure homogenizer at ≈1600 bar pressure for 30 cycles. Nanofibers were characterized by SEM which shows the average diameter of ≈66 nm. XRD analysis confirms the crystalline nature and a cellulose type-I structure of the nanofiber. In addition, obtained nanofibers were chemically surface modified by 12.91% of acetyl and 72.23% of carboxyl groups by varying reaction conditions.