Abstract <p>Cellulose nanocrystals (CNCs) were successfully extracted and purified from cotton fabric waste via alkali treatment and acid hydrolysis. The resulting nanostructure was characterized using transmission electron microscopy (TEM) and X-ray diffraction spectroscopy (XRD), confirming the successful isolation of CNCs. A PV/Cu²⁺-CNCs/CNFs/PVA hydrogel was then synthesized through chemical crosslinking of polyvinyl alcohol (PVA), cellulose nanofibrils (CNFs), cellulose nanocrystals (CNCs), and borax. The incorporation of CNCs as a reinforcing agent significantly enhanced the hydrogel’s surface area and water absorption capacity, leading to a substantial improvement in the colorimetric sensitivity of the sensor. This hydrogel was employed as a non-invasive colorimetric sensor for urinary cysteine detection, utilizing an indicator-displacement assay (IDA) with pyrocatechol violet/copper ion (PV/Cu²⁺) as the indicator. Upon exposure to cysteine, the sensor exhibited a distinct color change from greenish-blue to orange-red. The sensor demonstrated a linear detection range of 0–0.6 g/L and a limit of detection (LOD) of 0.017 g/L, effectively encompassing the clinical cut-off level for cysteine in human urine (0.25 g/L), relevant for Alzheimer’s disease indication. Eventually, this sensor was validated for cysteine determination in artificial urine samples, confirming its practical applicability.</p> Graphical abstract <p></p>