Fluorescently labeled cobalt peptide deformylase (Co-PDF) can be efficiently used as a fluorescence-resonance-energy-transfer-based sensing device for hydrogen sulfide (H,S). The proof of concept of our sensor system is substantiated by spectroscopic, structural, and theoretical results. Monohydrogen sulfide coordination to Co-PDF and Ni-PDF was verified by X-ray crystallography. Density functional theory calculations were performed to gain insight into the characteristics of the coordination adduct between H2S and the cobalt cofactor in Co-PDF.