Cu(II) can be reduced to Cu(I) by sodium ascorbate (SA) in situ, which in turn induces CuAAC reaction between the weak fluorescent compound (3-azido-7-hydroxycoumarin) and propargyl alcohol to form a strong fluorescent compound. Based on such principle, a simple and sensitive fluorescence sensor for Cu(II) can be developed, which combines the character of high selectivity of click chemistry and high sensitivity of fluorescence detection. The value of fluorescence increase factor shows a good linear relationship with the concentration of Cu(II) in the range of 0.25 μM-2.5 μM with a detection limit of 0.08 μM. In addition, the developed sensor shows high selectivity towards Cu(II) assay even in the presence of other common metal ions and it has been successfully applied to detect Cu(II) in human serum with satisfactory results.