In this work, a novel fluorescent sensor based on a quinoline derivative was designed and synthesized for detecting copper ions in a near-aqueous media. According to its extra-nuclear structure, copper (II) normally exhibits effectiveness for quenching the singlet excited state of organic chromophores through a fast electron transfer mechanism. However, this molecule displayed a strong fluorescence “turn-on” phenomenon upon addition of Cu2+, which was rarely reported in previous research. In addition, density functional theory calculations were adopted to investigate the molecular orbitals as well as the spatial structure. Furthermore, this sensor was applied into living SGC-7901 cells to extend its application in biological systems.