A novel “off-on” rhodamine B schiff fluorescent probe was synthesized by rhodamine B, hydrazine hydrate and Vanillin, it exhibits high selectivity for sensing Cu2 in 50% acetonitrile. The fluorescence titration spectra and the Job's plot indicated the formation of 1:1 complex between 1 and Cu2 , and the complexation process is reversible. For Cu(Ⅱ) concentration in the scope of 0～10 μmol/L, it showed a good linear relationship with a correlation of r=0.9972(n=5) and detection limit of 0.48 μmol/L.