Abstract：Objective：To evaluate the application of flow fluorescence assay(FFA) in detection of CEA, CYFRA21-1 and NSE. Methods：The precisions of FFA in the detection of CEA, CYFRA21-1 and NSE were measured. The correlations of the results between FFA and electrochemiluminescence immunoassay (ECLIA) in detection of CEA, CYFRA21-1 and NSE were determined. The reference intervals of CEA, CYFRA21-1 and NSE for the two methods were established. The accuracy of FFA for CEA, CYFRA21-1 and NSE was evaluated. Results：The intra assay CVs of FFA for CEA, CYFRA21-1 and NSE were less than 6%, and the inter assay CVs were less than 10%. The high correlation coefficients between FFA and ECLIA were found in the detection of CEA, CYFRA21-1 and NSE (rCEA=0.973 8, rCYFRA21-1=0.928 8, rNSE=0.982 0). The upper limits of reference interval for CEA, CYFRA21-1 and NSE were defined according to the first 95 percentile as P95 CEA=3.62 ng/mL, P95 CYFRA21-1=2.44 ng/mL and P95 NSE=11.23 ng/mL for FFA and P95 CEA=3.83 ng/mL, P95 CYFRA21-1=2.88 ng/mL and P95 NSE=14.85 ng/mL for ECLIA. Using ECLIA as the reference method, the coincidence rate of the two assays were 95.1% for CEA, 92.6% for CYFRA21-1, 93.68% for NSE, respectively. Conclusion：FFA for CEA, CYFRA21-1 and NSE showed accurate, fast and cost-saving. Three tumor makers could be detected simultaneously and small volume of sample was needed, so FFA should be worth applying for detection of the tumor markers.