Abstract: The analytical method for the determination of petroleum samples in water by fluorescence spectrophotometry was improved. The petroleum samples in water was extracted with n-hexane, purified by magnesium silicate, and detected using a fluorescence spectrophotometer. Under optimal conditions, the calibration curve of the method showed good linearity, the correlation coefficient was 0.999, and the detection limit was 0.007 mg/L. The method has been applied to determinate petroleum samples collected from different factory surface water and groundwater. The average spiked recoveries were among 87.5%~110.0%. Comparing ultraviolet and infrared methods with fluorescence spectrophotometry, the relative deviation of surface water and groundwater was 5.3%~13.3%, and the wastewater was 1.7%~19.5%. The improved method is accurate and sensitive, and can be used as a supplement, to the petroleum samples in water monitoring standard system.