We apply the localized surface plasmon resonance (LSPR) of gold nanoparticles (GNPs) covalently coupled with cytochrome c (cyt c) to create a nanobiosensor for detecting hydrogen sulfide (H₂S) in the range of 15–100 ppb. Monolayer formation of GNPs on glass surface functionalized with 3-aminopropyltrimethoxysilane (APTMS) is performed for fabricating a chip-based format of the optical transducer. By chemical introduction of short-chain thiol derivatives on cyt c protein shell via its lysine residues, a very fast self-assembled monolayer (SAM) of cyt c is formed on the GNPs. Significant shifts in the LSPR peak (Δλ_LSPR) are observed by reacting H₂S with cyt c. Results show a linear relationship between Δλ_LSPR and H₂S concentration. Furthermore, shifts in the LSPR peak are reversible and the peak positions return to their pre-exposure values once the H₂S is removed. The experimental results strongly indicate that the protein based LSPR chip can be successfully used as a simple, fast, sensitive and quantitative sensor for H₂S detection.