Linseed straw is the stalk or stem of the linseed plant that remains after the seeds have been harvested. This agricultural waste is generated every year and is mostly left or burned in fields, causing environmental problems. This study focuses on the isolation and characterization of crystalline nanocellulose (CNC) from linseed straw fibers as a potential alternative source. The CNCs were isolated using sequential chlorine-free chemical and mechanical treatments, involving three main steps: pre-treatment, acid hydrolysis, and post-treatment. The isolated CNC was thoroughly characterized for its morphology, particle size, aspect ratio, crystallinity, crystallite size, cellulose polymorph, thermal properties, and chemical composition, compared to raw fiber (RF) and micro cellulose (MC). Characterization techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FT–IR) were employed. The rod-like CNCs obtained exhibited a yield of 79.87 ± 1.35%, with a mean diameter of 7.06 ± 1.95 nm and a length of 66.14 ± 28.58 nm. The aspect ratio was measured at 10.02 ± 4.87 nm, with a crystallinity index of 73.29% and a crystallite size of 5.61 nm. Additionally, the CNCs displayed an average molecular weight of 2.36 × 104 g/mol, an average degree of polymerization of 146, and a peak decomposition temperature of 515°C. These results suggest that linseed straw fibers are a promising source for the production of CNC, which can effectively serve as a filler in polymer composites.