A rapid and quantitative up-converting phosphor technology-based later-flow assay (UPT-LF assay) was developed for on-site detection of Brucella. Different Brucella species both in pure cultures and in spiked samples could be quantitatively detected. The detection limit for pure culture was 5 x 10(6)CFU/ml and the sensitivity for different spiked samples ranged from 2.0 x 10(3) to 3.9 x 10(5)CFU/mg. The UPT-LF assay showed high specificity, reproducibility and stability, providing great potential for Brucella on-site detection.