Two novel similar structural coumarin-based fluorescent compounds L1 and L2 employing ether oxygen chain and 2 – pyridine unit had been prepared to explore the effect of structural adjustment on property. The prepared L2 through slightly adjusting structure of L1 exhibited the prominent fluorescence responses to Hg and Cu in HO, which could monitor the corresponding metals ion in mitochondria of Hela cells, but L1 only recognized Hg through turn-on fluorescence response in CHOH/HO, fluorescence co-localization studies illustrated that the L1 mainly located at lysosome. The recognition mechanism had been established by H NMR titration, Job's plot, MAlDI-TOF mass spectrometry and single crystal structure. Bio-imaging experiments revealed that L1 and L2 could qualitatively monitor Hg or Cu in Hela cells and mouse kidney tissues.