An up-converting phosphor technology-based biosensor (UPT-based biosensor) was developed for immunoassay and fast detecting using up-converting phosphor (UCP) as the biological marker. UCP can emit visible light when excited by infrared light. Through detecting and analyzing the content of UCP particle on the test strip after immunoreaction, the UPT-based biosensor can obtain the concentration information of target analyte in the sample. The UPT system realized the quantitative detection and had good ability to meet the need of some emergencies. High sensitivity (nanogram/ml), good linear relationship in the concentration range of 200～6000 ng/ml and an excellent correlation (R2≥0.95) were verified by quantitative detection results. The sensitivity of the UPT-based biosensor was better than that of the indirect hemagglutination test in the practical application. All the results were comparable with that obtained by Western Blot detection. The UPT-based biosensor had stable, reliable and sensitive working performances, and it can meet the need of some various applications such as rapid immunoassay, chemical and biological detection and so on.