Quantitative analysis of chlorobenzene (CB) in water by three-dimensional fluorescence spectrometry was discussed in the present paper. The study showed that there is only one fluorescence peak for CB which lies in the range of excitation wavelength (λex) 210～240 nm and emission wavelength (λem) 330～370 nm. When measuring CB solution of concentration 0.002~0.05 mg·L-1, the fluorescence intensity was the strongest as λex/λem was 225/340 nm, which presented linear correlation with concentration, and the related coefficient was 0.999 67. The study proved that three-dimensional fluorescence spectrometry can be adopted for quantitative analysis of CB in water. With this method, the detection limit was 3.68×10-6 mg·L-1 and the standard deviation was 0.04% at 90% confidence level.