A simple and sensitive method for the simultaneous determination of trace amounts of 1-naphthol and 2-naphthol by fluorescence spectrofluorimetry was established based on the fact that the excitation and emission wavelengths are different between 1-naphthol and 2-naphthol. The influencing factors, such as pH temperature and surface activator were synthetically considered in the present paper. Interference of similar organic matters and inorganic ions were also taken into account. Compared with traditional methods, the method discussed here needs no complex pretreatments and expensive equipment. So it is convenient, quick and cheap yet with a satisfactory precision and accuracy. The accuracy and precision of results obtained are satisfactory when the concentration ratio (CR) of 1-naphthol to 2-naphthol is in the range of 1:19-19:1; the detection limit (DL) of both naphthols is 8.6 x 10(-3) ng x mL(-1); the relative standard deviation (RSD) of 1-naphthol and 2-naphthol is 1.26% and 1.45% respectively; and the recovery ratio (RR) of both naphthols is between 95% and 105%. This proposed method has been applied to the determination of trace amounts of 1-naphthol and 2-naphthol in the man-made sample water and environmental imitative water simultaneously, and obtained a satisfactory result, showing that the method discussed here is simple, sensitive and reliable in the future use.