A method of high performance liquid chromatography-fluorescence spectroscopy (HPLC-FLD) was used to determinate L-tryptophan in urine of Acipenser sinensis and A. dabryanus. The samples were extracted by methanol and acetonitrile, by centrifugation to get supernatant, and purified by an ENVI-18 solid-phase extraction column. The acetonitrile-1% (volume fraction) formic acid aqueous solution (10:90, volume fraction) was used as the mobile phases, the extracts were separated on a symmetry@C18 chromatographic column (250 mm×4.6 mm, 5 μm). The samples were determinated under the condition of the flow rate of 1.0 mL/min, with the fluorescence wavelength of λ_ex=285 nm, λ_em=353 nm. The results indicated that the significant linear relationships between peak areas and mass concentrations of the analytes were obtained in the range of 0.010-2.000 μg/mL (R²=1), and linear regression equation was y=247.27x-0.125 5. The limits of detection (LOD) and limits of quantification (LOQ) of L-tryptophan were 0.005 μg/mL and 0.020 μg/mL. The recoveries of blank matrix and sample substrate with standard samples were 82.2%-102.1% and 85.6%-95.1%, respectively, with less than 5.7% of the relative standard deviations (RSD). This method has some obvious advantage such as easy operation, time saving, high recoveries and high accuracies. It will be valuable to analyze L-tryptophan in urine of A. sinensis and A. dabryanus. A methodology basis is provided to study the role of L-tryptophan in information exchange of A. sinensis and A. dabryanus.