The rising antimicrobial resistance is resulting in increased failure rates of Helicobacter pylori (H. pylori) eradication therapy in clinical settings, and there is an urgent need to develop novel selective therapeutics independent of antibiotics. Superoxide dismutase (SOD) is an indispensable player in the survival, colonization, pathogenicity, and antimicrobial resistance of H. pylori. Based on the heterologous expression of H. pylori 26,695 SOD (HpSOD), this study designed and constructed an HpSOD-based biosensor through a three-step cascade reaction strategy, successfully identifying four potential HpSOD inhibitors. Among them, nitrilotriacetic acid trisodium salt exhibited the strongest intrinsic binding affinity for HpSOD, while 4,5-dichloro-2-(3-methylphenyl) pyridazin-3(2H)-one demonstrated the most significant apparent inhibitory efficacy against HpSOD. Meanwhile, 4-chloro-3,5-dinitrobenzoic acid, nitrilotriacetic acid trisodium salt, and deferoxamine did not inhibit human SOD in vitro, demonstrating excellent selectivity. Moreover, molecular docking revealed the characteristics of the catalytic center and the allosteric regulatory site of HpSOD. This study provides a theoretical basis and novel strategies for the treatment of H. pylori infection based on the inhibition of HpSOD activity.