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Bladder cancer is a heterogeneous disease and occupies highest incidence in developed country. Time-resolved fluoroimmunoassays (TRFIA) is a new detection technique with a feature of sensitive, simple and inexpensive. The aim of this study is to establish a dual-label TRFIA for the simultaneous detection of BLCA-4 and NMP52 in urine in a single run. The sandwich immunoassay was used to detect the concentration of BLCA-4 and NMP52 in urine. BLCA- 4 and NMP52 in urine were captured by anti-BLCA-4 and anti-NMP52 antibodies immobilized on microtiter wells, and then banded together with another anti-BLCA-4 and anti-NMP52 labeled with europium(III) Sm3+ and samarium(III) Eu3+ chelates, followed by fluorescence measurement using time resolved fluorometry. To assess the performance of this assay, clinical urine samples were used, and then commercialized kits were compared. The sensitivity for BLCA-4 and NMP52 detection of this assay were 2 U/mL (dynamic range, 5-300 U/L) and 1 μg/ml (dynamic range, 2-150 μg/ml) respectively. The correlation coefficients (R) between the present dual-label TRFIA and commercially available kits were high. R-values were 0.99 for BLCA-4 and NMP52. The cross-reactivity seems not to influence the results. The present dual-label TRFIA, allowing the simultaneous detection ofBLCA-4 and NMP52, has high sensitivity, specificity, and accuracy in clinical sample analysis. Therefore, it has good prospects of application.