Abstract: In present study, cellulose nanowhiskers (CNWs) were isolated from roselle fibers by employing low-medium amplitudes of ultrasonication. In a range of low-to-moderate amplitudes of ultrasound, 20%, 30% and 40% amplitudes were applied during ultrasonication treatment to produce CNW-I, CNW-II and CNW-III particles, respectively. The morphological (TEM, FESEM, and AFM), physicochemical (FTIR, EDS, DLS, and XRD) and thermal properties (TGA and DSC) of produced CNWs were conducted to understand the effect of applied amplitudes on CNWs properties. It is clear from the FTIR spectra that increasing ultrasonic amplitudes enhanced crystalline of CNWs. In TEM analysis, CNWs sonicated with 30% and 40% amplitudes possessed the shape of elongated rod-like nanoparticles. FESEM and AFM micrographs exhibited varying whisker-like nanostructures. Additionally, both CNW-II and CNW-III showed stable aqueous colloidal suspensions with zeta potential values more than − 25 mV in response to high sulfur content. As for XRD evaluation, CNW-III exhibited the higher crystallinity degree of 79.9% amongst the all samples. Based on thermal analysis, CNW-I and CNW-II possessed high heat resistant capability at elevated temperature. These CNWs are potential reinforcements in nanocomposites for diverse applications in packaging, engineering, composites and biomedical fields.