Abstract: A new method for the simultaneous determination of tyrosine and tryptophan by polarization spectrofluorimetry has been developed. The interference of phenylalanine and light scattering of solvent were eliminated by using vertically crossed polarizers. The overlapping of fluorescence spectra of the two amino acids was solved with filters. The linear ranges of determination were 0.02～12.0 mg·L^-1 (tyrosine) and 0.01～2.50 mg·L^-1 (tryptophan). The mean recoveries of tyrosine and tryptophan in samples were 95.4% and 101.7% (in amino acid injection). 99.2% and 100.0% (in wild jujube kernel), 95.3% and 97.2% (in shrimp shell) respectively. The proposed method has been used to determine tyrosine and tryptophan in amino acid injection, extracts of wild jujube kernel and shrimp shells with satisfactory results.