In order to realize real-time monitoring of Cu2+, a new fluorescent probe HL, a Schiff base derivative of N-n-butyl-4-[2]-1,8-naphthalimide, has been designed and synthesized. In methanol-HEPES [2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid] solution (1:1, v/v, pH = 7.4) HL showed excellent selectivity towards Cu2+ over other common coexisting metal ions. The fluorescence intensity for HL showed a good linearity with the concentration of Cu2+ ions in the range of 0.5–5.0 μm. Based on combined fluorescence titration, Job’s plot analysis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry results, Cu2+ forms a 1:2 complex with L. The fluorescence intensity of HL exhibits significant quenching after binding with Cu2+, owing to the strong, intrinsic paramagnetic behavior of Cu2+. Ultimately, in order to test the performance of the synthesized probe, HL was preliminarily applied to the determination of Cu2+ in the Yellow River and in tap water with satisfying results.