Detection of selenocysteine (Sec) content in cells by fluorescence probe is of great significance for the identification of human related diseases. To achieve fast and sensitive detection of Sec, two isomers A4 and B4 as turn-on fluorescent probes to detect Sec were designed and synthesized. Both A4 and B4 display fast turn-on response, high selectivity and sensitivity toward Sec, which can be applied for fluorescence imaging of Sec in living cells. Compared with B4, A4 has a larger Stokes shift (125 nm), wider pH range (5-10) and lower detection limit (65.4 nM) due to its ESIPT (excited state intramolecular proton transfer) effect. In view of the detection performance of these two probes, they can be used as effective tools for detecting Sec in biological systems.