This study aimed to establish a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. Some Cystatin-c immunoassays are sensitive, accurate, and available for clinical application, but they are expensive and time-consuming procedures. Also, conventional organic dye-based fluorescence lateral flow assay showed more background fluorescence interference and low analytical sensitivity. So this Europium-based sandwich immunoassay was developed to detect the concentration of cystatin-c in a urine sample with captured anti-cystatin-c antibodies immobilized on nitrocellulose membrane and then bonded with detection anti-cystatin-c labelled with CM-EU, followed by fluorescence measurement using time-resolved fluorometry in 15 minutes. The performance of this TRFIA was evaluated using the clinical urine serum and compared with the ELISA assays. The linear calibration range was 0.015-32 µg/ml, and the limit of detection (LOD) quantified was 0.0001µg/ml. This current work has improved the LOD of our previous work from 0.013µg/ml to 0.001µg/ml. These results indicated that the CM-EU nanoparticle-based LFIA is rapid, more sensitive, reliable, and reproducible for point-of-care testing of Cys-C concentrations in urine