Background: A rapid and sensitive time-resolved fluoroimmunoassay (TRFIA) based on the biotin-streptavidin amplification system was developed for the determination of anticyclic citrullinated peptide (anti-CCP). Methods: Europium-labeled streptavidin derivatives combined with europium and anhydride of diethylene triamine pentaacetic acid were used to label streptavidin, biotin was coupled with rabbit antihuman IgG to form a biotin-anti-human IgG bridge between streptavidin-europium and the anti-CCP antibody in the immunoassay. The anti-CCP assay was carried out by measuring the fluorescence of Eu3+-streptavidin at 615 nm. Results: The presented method produced a wide linear range from 0.58 to 9,463 U/ml, while it was only 591.4-18.48 U/ml when using an ELISA kit, and featured a detection limit up to 0.5 U/ml for anti-CCP. The values determined by the biotin-streptavidin-TRFIA and ELISA correlated well (R-2 = 0.8927). The method was applied to determine anti-CCP in serum samples with satisfied recoveries of 96.45-104.63%. Conclusion: The assay results obtained by the present method showed that biotin-streptavidin-amplified TRFIA improve the traditional ELISA kit for anti-CCP detection. Therefore, it offers a better alternative immunoassay in rheumatoid arthritis management. (C) 2014 Wiley Periodicals, Inc.