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 Hg2+ and Cu2+ in H2O, which could monitor the corresponding metals ion in mitochondria of Hela cells, but L1 only recognized Hg2+ through turn-on fluorescence response in CH3OH/H2O, fluorescence co-localization studies illustrated that the L1 mainly located at lysosome. The recognition mechanism had been established by H-1 NMR titration, Job's plot, MAlDI-TOF mass spectrometry and single crystal structure. Bio-imaging experiments revealed that L1 and L2 could qualitatively monitor Hg2+ or Cu2+ in Hela cells and mouse kidney tissues.