A simple and cost-effective fluorescence immunoassay for the sensitive quantitation of disease biomarker α-fetoprotein (AFP) has been developed based on the phosphate-triggered fluorescence turn-on detection of alkaline phosphatase (ALP), with the reversible binding between calcein and Ce3+ as a signaling element. In this immunoassay, fluorescent calcein is readily quenched by Ce3+ via a coordination process. The ALP-catalyzed hydrolysis of p-nitrophenyl phosphate leads to the formation of p-nitrophenol and inorganic orthophosphate, and the newly formed orthophosphate could potently combine with Ce3+ due to the higher affinity, thus, recovering the fluorescence of calcein. The corresponding fluorescence signal triggered by phosphate is related to ALP activities labeled on antibody, and thus could be applied to detect target antigen in an enzyme-linked immunosorbent assay (ELISA) platform. The fluorescence intensity correlated well to the AFP concentration ranges of 0.2-1.0 and 1.0-4.0 ng/mL, with a detection limit of 0.041 ng/mL. The proposed fluorescence ELISA possesses convincing recognition mechanism and exhibits excellent assay performance in the evaluation of the AFP level in serologic test, which unambiguously reveals great application potential in the clinic diagnosis of disease biomarkers.