In this study, cellulose nanofibers (CNFs) were successfully isolated from rapeseed straw (RS) whose valorization has been rarely investigated to date. A combined bleaching method without chlorine was applied for the purification of cellulose fibers, previously unexplored for RS. Chemical composition analysis and Fourier-transform infrared spectroscopy (FTIR) indicated that the purification method eliminated hemicellulose and reduced lignin content from 24.4 % to 1.8 %. The isolation of CNFs was performed using sulfuric acid hydrolysis under different acid concentrations (55 and 60 % v/v) and hydrolysis times (15, 30, and 45 min). The isolated CNFs were characterized by FTIR, X-ray diffraction (XRD), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). The formation of CNFs was confirmed by a significant increase in crystallinity index from 46.45 % of RS to >79.41 % of CNFs, depending on acid concentration and isolation duration. Carboxymethyl cellulose (CMC) films with different contents of CNFs were prepared by casting method. The mechanical properties and cytotoxicity of the prepared films were investigated. The CNFs obtained from RS via a chlorine-free purification method showed promising results for their usage as reinforcement in CMC matrix and film fabrication for various applications such as transdermal medicine and food packaging.