A novel turn-off strategy was developed for the melamine (Mel) detection based on melamine-Cu conjugate and subsequent interrupted the formation of AT-dsDNA copper nanoparticles (AT-dsDNA CuNPs). The coordination of melamine to copper cannot make the reduction of Cu2+ to Cu0 enough in the AT24-CuNPs synthesis process, and thereby results in the fluorescence intensity of CuNPs decreasing. Under the optimum conditions, the concentration of melamine showed a good linear relation with the fluorescence response of CuNPs in the range of 1~150 μmol·L-1. The detection limit was 0.5 μmol·L-1. Furthermore, good recoveries were obtained while the proposed method was applied to the analysis of melamine in milk samples.