The mechanism of interaction of trypsin with the sulfathiazole was studied through using fluorescence quenching and UV-visible absorption spectra at pH 7.4. The Stern-Volmer quenching constants, binding constants, number of binding sites and the corresponding thermodynamic parameters Delta H-o, Delta S-o and Delta G(o) were calculated at different temperatures. The effect of common metal ions on the constants was also discussed. The results suggest that sulfathiazole can interact strongly trypsin and that there is the formation of trypsin-sulfathiazole complex and the interaction can be explained on the basis of hydrogen bonds and van der Waals forces. The binding distance (r) between the donor (trypsin) and acceptor (sulfathiazole) was 3.52 nm based on the Forster's non-radiative energy transfer theory. The detection and quantification limits of sulfathiazole were calculated as 2.52 and 8.40 mu M in the presence of trypsin, respectively. The relative standard deviation (RSD) was 4.086 % for determinations (n = 7) of a sulfathiazole solution with the concentration of 7.54 mu M.