Background:　Gold　nanoclusters　(AuNCs)　obtained　by　electroreduction　have　excellent　electrochemiluminescence　(ECL)　properties,　and　its　ECL　intensity　is　regulated　by　the　valence　state.　In　addition,　their　ECL　signals　can　be　rapidly　quenched　by　reactive　oxygen　species　(ROS).　Based　on　this　observation,　a　sensitive　ROS　biosensor　was　designed　based　on　valence　regulation　of　AuNCs.　Excessive　ROS　in　seminal　plasma　can　lead　to　male　infertility,　and　the　short　half-life　and　instability　of　ROS　pose　a　challenge　for　their　detection.　Since　valence　regulation　can　be　done　quickly　and　is　very　sensitive,　this　ECL　biosensor　holds　promise　to　address　this　issue.　Results:　The　ECL　mechanism　of　AuNCs　and　the　quenching　mechanism　of　AuNCs　by　ROS　were　explored,　mainly　because　ROS　change　the　valence　state　of　AuNCs.　The　ECL　signals　of　the　biosensor　have　a　linear　relationship　with　logarithm　of　the　target　concentration　in　the　range　of　1.0　x　10(-8)　to　1.0　x　10(-3)　M　and　1.0　x　10(-3)　to　1.0　x　10(-1)　M,　with　a　detection　limit　of　0.75　x　10(-10)　M　(S/N　=　3).　The　biosensor　enables　rapid　one-step　detection　of　ROS　and　has　the　advantage　of　being　stable　and　reusable.　More　notably,　the　results　of　57　real　samples　showed　that　the　biosensor　can　be　used　to　accurately　assess　the　concentration　of　seminal　plasma　ROS,　guiding　the　monitoring　of　sperm　quality　and　the　diagnosis　of　male　infertility.　Significance:　Compared　with　the　traditional　strategy　of　applying　AuNCs　only　as　a　luminescent　body,　this　strategy　of　regulating　the　valence　state　of　AuNCs　to　achieve　sensitive　and　rapid　detection　broadens　the　application　of　AuNCs　in　the　field　of　analysis.　Compared　with　other　ROS　detection　strategies,　the　one-step　immediate　detection　method　effectively　avoids　the　inaccuracy　caused　by　the　short　half-life　and　natural　dissipation　of　ROS,　and　is　expected　to　improve　the　accuracy　and　efficiency　of　clinical　diagnosis.