Two new twinborn benzimidazole derivates (L and A), which bonded pyridine via the ester space on the opposite and adjacent positions of the benzene ring of benzimidazole respectively, were designed and synthesized. Compound L displayed fluorescence quenching response only towards copper(II) ions (Cu 2+ ) in acetonitrile solution with high selectivity and sensitivity. However, compound A presented 'on-off' fluorescence response towards a wide range of metal ions to different degrees and did not have selectivity. Furthermore, compound L formed a 1:1 complex with Cu 2+ and the binding constant between sensor L and Cu 2+ was high at 6.02 × 10 4 M −1 . Job's plot, mass spectra, IR spectra, 1 H-NMR titration and density functional theory (DFT) calculations demonstrated the formation of a 1:1 complex between L and Cu 2+ . Chemosensor L displayed a low limit of detection (3.05 × 10 −6 M) and fast response time (15 s) to Cu 2+ . The Stern-Volmer analysis illustrated that the fluorescence quenching agreed with the static quenching mode. In addition, the obvious difference of L within HepG2 cells in the presence and absence of Cu 2+ indicated L had the recognition capability for Cu 2+ in living cells.