Hydrogen　sulfide　(H2S)　in　joint　fluid　acts　as　a　signal　molecule　to　regulate　joint　inflammation.　Direct　detection　of　H2S　in　joint　fluid　is　of　great　significance　for　the　diagnosis　and　treatment　of　arthritis.　However,　due　to　the　low　volume　of　joint　fluid　and　low　H2S　concentration,　existing　methods　face　the　problem　of　the　insufficient　limit　of　detection.　In　this　study,　a　highly　sensitive　biosensor　was　proposed　by　designing　a　primer　probe　and　combining　it　with　hybrid　chain　reaction　(HCR)　under　the　strong　interaction　between　metal　ions　and　H2S　to　achieve　H2S　detection.　The　primer　probe　containing　multiple　cytosine　(C)　sequences　was　fixed　on　a　gold　electrode,　and　the　C–Ag–C　hairpin　structure　was　formed　under　the　action　of　Ag+.　In　the　presence　of　H2S,　it　can　combine　with　Ag+　in　the　hairpin　structure　to　form　Ag2S,　which　leads　to　the　opening　of　the　hairpin　structure　and　triggers　the　hybridization　chain　reaction　(HCR)　with　another　two　hairpin　structures　(H1　and　H2).　A　large　number　of　double‐stranded　nucleic　acid　structures　can　be　obtained　on　the　electrode　surface.　Finally,　Ru(phen)32+　can　be　embedded　into　the　double　chain　structure　to　generate　the　electrochemiluminescence　(ECL)　signal.　The　linear　response　of　the　H2S　biosensor　ranged　from　0.1000　to　1500　nM,　and　the　limit　of　detection　concentration　of　H2S　was　0.0398　nM.　The　developed　biosensor　was　successfully　used　to　determine　H2S　in　joint　fluid.　©　2022　by　the　authors.　Licensee　MDPI,　Basel,　Switzerland.