Half of patients with renal cell carcinoma (RCC) develop metastases. New and noninvasive biomarkers are needed for the diagnosis of RCC. The study aims to develop an EV-based assay for the detection of RCC using a highly sensitive nanoparticle-aided time-resolved fluorescence immunoassay (NP-TRFIA). To confirm the presence of tetraspanins on EVs, size exclusion chromatography is used to separate EV- and PE-fractions from RCC4, 786-O, and HEK293 cell lines. EV- and PE-fractions are quantified using NP-TRFIA assays established for CD9, CD63, CD81, and CD151. Tetraspanins are measured from RCC CCM and serum samples of RCC (n = 14), benign (n = 17), and healthy (n = 9) individuals. Among the tetraspanins, CD63 exhibits 3-5-fold higher expression on RCC4 and 786-O CCM compared to HEK293. A sandwich CD63-CD63 assay demonstrates significant discrimination of RCC patients from benign (p = 0.0003), and healthy (p = 0.005) individuals, respectively. Similarly, the CD81-CD81 assay also enables significant separation of RCC patients compared to benign (p = 0.014), and healthy (p = 0.003) controls, respectively. This suggests that RCC cell lines and serum of RCC patients show higher amounts of CD63- and CD81-EVs compared to controls. Detection of these EVs using NP-TRFIA approach may play a vital role in the detection of RCC.