In Britton-Robinson (BR) buffer solution at pH 4.0, erythrosine (ET) reacts with tetracaine hydrochloride (TA x HCl) to form an ion association complex that quenches the fluorescence of erythrosine and has a compound ratio of 1 : 1. The measurement wavelength of fluorescence quenching was located at lambda(ex)/lambda(em) = 525 nm/556 nm. The linear range for TA x HCl is from 0.28 microg x mL(-1) to 4.8 microg x mL(-1), and its detection limit is 0.083 microg x mL(-1). The influence of coexisting substance was also inspected, and the result shows that this method has a better selectivity. Therefore, a new fluorospectrophotometry, being high sensitive, sample and rapid, was developed to determinate TA x HCl at trace amounts. The method has been applied to the determination of TA x HCl in human serum and urine samples with satisfactory results. In this study, the spectral characteristics and the optimum reaction conditions were discussed with quantum chemistry AM1 method.