4-Bis-(2-hydroxyethyl)amino-N-n-butyl-1,8-naphthalimide was developed into a turn-on fluorescent sensor for Cr(VI). In the mixture of N,N-dimethylformamide and deionized water (1:9, v/v), the sensor could highly selectively distinguish CrO4(2-) from Cr(3+), Na(+), K(+), Mg(2+), Ca(2+), Mn(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), NO(3-), SO4(2-), PO4(3-), IO3(-), ClO(-) and Cl(-) by a 6-fold fluorescence enhancement. The working concentration of CrO4(2-) was from 0 to 90 μM with a detection limit of 3.6 × 10(-7) mol/L. The detection could be carried out in water matrix and within a wide pH range (approximately from 2 to 12), and a large number of environmentally and biologically relevant ions including Cr(3+) showed no significant interferences with the detection. The sensing mechanism was explored by reversibility and LC/MS analysis, as well as Job's plot experiment, and the results suggested that the recognition was based on the oxidation of the primary alcohol in the structure of the sensor by the Cr(VI) sources.