A new highly selective and sensitive fluorescent probe for Cu2+, N-n-butyl-4-(1′-cyclooctene-1′,3′,6′-triazole)-1,8-naphthalimide (L), was synthesized and evaluated. The structure of compound L was characterized via IR, 1H-NMR, 13C-NMR and HRMS. The fluorescent probe was quenched by Cu2+ with a 1:1 binding ratio and behaved as a “turn-off” sensor. An efficient and sensitive spectrofluorometric method was developed for detecting and estimating trace levels of Cu2+ in EtOH/H2O. The ligand exhibited excitation and emission maxima at 447 and 518 nm, respectively. The equilibrium binding constant of the ligand with Cu2+ was 1.57 × 104 M−1, as calculated using the Stern-Volmer equation. Ligand L is stable and can be used to detect Cu2+ in the range of pH from 7 to 12. The sensor responded to Cu2+ rapidly and a large number of coexisting ions showed almost no obvious interference with the detection.