The surfactant sensitized spectrofluorimetry for speciation of chromium (Cr(VI)/Cr(III)) was developed. The analytical procedure was that the fluorescence intensity of l4,10,16,22-tetramethoxyl resorcinarene carboxylic acid derivatives (TRCA) could be selectively quenched by Cr(VI) and the fluorescence quenching value (ΔF=FTRCA-FCr(VI)-TRCA) was increased in cetyltrimethylammonium bromide (CTAB). The main influence factors on the fluorescence quenching (ΔF) were investigated in detail. Under the optimal conditions, the linear range of calibration curve for the determination of Cr(VI) was 0.10~5.00 μg/mL, and the detection limit was 0.024 μg/mL with RSD=2.10% (c=1.0 μg/mL, n=3). The concentration of Cr(III) was calculated by subtracting Cr(VI) from the total chromium determined after oxidizing Cr(III) to Cr(VI). The preliminary sensitized mechanism was discussed with the inclusion constant (K) of TRCA-Cr(VI), the fluorescence quantum yield of TRCA, and IR spectra characterization. The method has been applied to the speciation analysis of Cr(VI)/Cr(III) in water samples.