A simple and sensitive probe, pyridine-based Schiff base (PBSB), is prepared based on the previous studies, which shows a rapid response to copper (II) through a fluorescence enhancement process. On association with copper (II), PBSB immediately presents a large red-shift (130 nm) in emission spectrum simultaneously accompanying with increasing emission intensity. The mechanism and binding sites of PBSB detecting copper (II) have been established by theoretical calculations and 1HNMR study, respectively. More importantly, in the complex biological system, PBSB having an excellent membrane permeability and a great photostability can visualize copper (II) in lipid droplet by bio-imaging, which display so great promising advantages in the issues causing by disorder of copper ions that could be convenient for prevention and control problems with human health and complex bio-system.