This work implies the preparation, structural and fluorescent characterization, and application of the 1,8-naphthalimide-based fluorescent probe for HS detection in human blood serum. The mechanism of fluorescent detection of HS is based on the reduction of the azide group, as the chemical reactive site, in the fluorescent probe structure, to the amino group. Compound 2 possesses another important structural motif, ethanolamine moiety, which modulates physicochemical properties. The synthetic pathway for the preparation of compound 2 is consisted of two reaction steps, and the target compound was prepared with a yield of 56%. The prepared compound 2 was characterized using H and C NMR spectroscopy and elemental analysis. During the fluorescence spectra measurements, several influences on fluorescence intensity were investigated, including pH, time dependence, selective response, and influence of HS concentration. The detection limit was calculated, which was 0.16 μmol L. For examination of the application of compound 2 for HS detection in real samples, compound 2 was successfully applied for HS detection in human blood serum. The concentration of HS in the human serum sample was 16.2 μmol L.The accuracy of HS determination in human blood serum samples is confirmed by the standard addition method and UV-Vis spectrophotometry method using methylene blue.