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 (HNMR)-H-1 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.