Abstract A simple, rapid, and sensitive spectrofluorimetric technique for the microdetermination of epinephrine in human serum is described. The investigation shows that trace amounts of epinephrine, antidepressant of clinical importance, can be determined without the conventional derivatization or use of fluorophores by diazotization. The method is based on the optimization of experimental parameters, such as pH, temperature, careful selection of excitation and emission wavelengths and on the use of anionic surfactant, sodium dodecyl sulfate (SDS), to enhance sensitivity. The measurement was carried out at 360 nm with excitation at 286 nm. Under the optimum conditions, a linear relationship was obtained between the fluorescence intensity and epinephrine concentration in the range of 0.10 and 1.0 μg/mL; the correlation coefficient and detection limit are 0.9953 and 0.05 μg/mL, respectively. Recovery tests indicated an efficiency of 95.5–98.7% by using known amounts of epinephrine spiked with human serum.