Accurate sensing of small biomolecules from complex biofluid media remains a challenge due to adverse interaction of metallic salts and other biofluid components. L-cystine is the most important biomarker for the disease cystinuria. Recognition of L-cystine in urine is thus fundamental for the timely detection of cystinuria and related chronic kidney disease (CKD). Poor solubility of L-cystine offer limits to design suitable sensors. Herein, we report the synthesis of thioglycolated-β-CD (TG-β-CD) anchored silver nanoparticles (AgNP) as a potential receptor (TG-β-CD⊃AgNP) for the effective optical recognition and quantification of L-cystine through UV–Vis and SERS (surface enhanced Raman spectroscopy) spectroscopic fingerprinting and suitable colourimetric naked-eye detection of L-cystine by indicator displacement assay. Methyl orange (Met-O) indicator was precomplexed with the receptor to generate the probe solution (Met-O@TG-β-CD⊃AgNP). Indicator has been exchanged from Met-O@TG-β-CD⊃AgNP by the addition of L-cystine, with an instantaneous visible colour change from yellow to red. The method is tolerable to other interfering abundant ions and biomolecules. Based on the innovative sensing assay a cellulose paper-dye test strip is developed for point-of-care detection and quantification of the biomarker. Structures of TG-β-CD, TG-β-CD⊃AgNP and Met-O@TG-β-CD⊃AgNP were elucidated by UV–Vis, FT-IR, PXRD, 1H NMR, etc. spectroscopy. Scanning electron microscopy (SEM) was used to examine the morphology of TG-β-CD, AgNP and TG-β-CD⊃AgNP. The chemical changes during the assay were evaluated by conductance, UV–Vis and SERS. The competitive displacement of the indicator and UV turn-on at ppm level analyte concentration made the process compatible for day-to-day point-of-care units.