This study aimed to establish a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. An Europium based Time resolved fluorescence immunoassay was developed to detect the concentration of Cystatin-C in a urine sample to increase the sensitivity 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 min. 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 0.0001 µg/ml. This current work has improved the LOD of our previous work from 0.013 µg/ml to 0.001 µg/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. © 2022, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.