Utilizing the dramatic fluorescence difference between the P 2 O 7 :Ce,Tb and PO 4 :Ce,Tb nanoparticles, we proposed the onestep optical strategy for fast, sensitive identification of alkaline phosphatase (ALP) in the present work. In this strategy, the pyrophosphate (PPi) combined with the rare earth ions Ce 3+ , Tb 3+ for its high affinity to metal ions to generate P 2 O 7 :Ce,Tb nanoparticles, which exhibit the bright characteristic green emission of Tb 3+ , it origins from the efficient energy transfer from Ce 3+ to Tb 3+ , the whole process can almost be finished instantaneously. While in the presence of ALP, the enzyme substrate PPi can be broken down into PO 4 3− , which can also self-assemble with Ce 3+ , Tb 3+ ions to produce PO 4 :Ce,Tb nanoparticles, but with a much weaker green light of Tb 3+ , and the greater ALP concentration, the weaker green light can be obtained. Based on this, we proposed a facile optical platform for ALP sensing, compared with those reported probes, it avoids the burdensome process of nanoparticle synthesis and target measurement. The experiment result shows that the fluorescence intensity of P 2 O 7 :Ce,Tb at 545 nm is linear to the concentration of ALP in a range of 0.25−250 U/L, and the detection limit is calculated to be 0.18 U/L. In addition, the excellent selectivity of the present strategy has been confirmed as well; the ALP activity suppression and the real serum sample test demonstrate its outstanding reliability forcibly.