Alkaline phosphatase (ALP) has been known to participate in the multiple pathways of cell metabolism. Here, a ratiometric fluorescent probe was designed for determination of ALP activity based on [email protected] The [email protected] nanoparticles were prepared via the self-assembly of guanosine monophosphate (GMP) with terbium and cerium ions (Ce3+, Tb3+). The [email protected] nanoparticles own two luminescent centers, Tb and Ce, respectively. In presence of ALP, the phosphate ester in GMP was hydrolyzed and the structure of [email protected] was destroyed. Moreover, the coordination effect of Ce3+, Tb3+ and ligand GMP was significantly declined and the energy transfer efficiency from Ce3+, GMP to Tb3+ was reduced. Therefore, the fluorescence emission of Tb3+ and Ce3+ was quenched and the fluorescence intensity ratio (F552/F384) gradually decreased. This probe displayed low limit detection 0.12 mU/mL under the concentration range of 0.2–60 mU/mL. Meanwhile, the method accomplished the detection of ALP in a diluted serum sample of 5%. This strategy will make a construction for the application of fluorescent probes in bioanalytical sciences.