Hydrogen sulfide (H₂S) has attracted attention in biochemical research because it plays an important role in biosystems and has emerged as the third endogenous gaseous signaling compound along with nitric oxide (NO) and carbon monoxide (CO). Since H₂S is a kind of gaseous molecule, conventional approaches for H₂S detection are mostly based on the detection of sulfide (S2-) for indirectly reflecting H₂S levels. Hence, there is a need for an accurate and reliable assay capable of determining sulfide in physiological systems. We report here a colorimetric, economic, and green method for sulfide anion detection using in situ formation of silver nanoparticles (AgNPs) using dopamine as a reducing and protecting agent. The changes in the AgNPs absorption response depend linearly on the concentration of Na₂S in the range from 2 to 15 μM, with a detection limit of 0.03 μM. Meanwhile, the morphological changes in AgNPs in the presence of S2- and thiol compounds were characterized by transmission electron microscopy (TEM). The as-synthetized AgNPs demonstrate high selectivity, free from interference, especially by other thiol compounds such as cysteine and glutathione. Furthermore, the colorimetric sensor developed was applied to the analysis of sulfide in fetal bovine serum and spiked serum samples with good recovery.