Cetyl trimethyl ammonium bromide (CTAB) stabilized perylene nanoparticles (PNPs) were prepared by a modified reprecipitation method in aqueous solution under ultrasonic treatment. A spectrofluorimetric method for the quantitative determination of hexavalent chromium (Cr(VI), dichromate species) based on the fluorescence (FL) quenching of CTAB-stabilized PNPs (CTAB-PNPs) in aqueous solution was proposed. Under the most favourable conditions, the FL intensity of PNPs monitored at an excitation wavelength of λ_ex = 382 nm was quenched by the successive addition of increasing concentrations of dichromate ions. The FL quenching results were found to fit the Stern–Volmer (S–V) relationship in the range of 0.5–50 μg mL⁻¹ with a correlation coefficient of 0.9997. The limit of detection (LOD) was 0.008 μg mL⁻¹. The method based on FL quenching was successfully applied for the quantitative analysis of Cr(VI) in water samples collected from different environments.