The acidic environment of the stomach is a threat to the curative effect of antimicrobial drugs for the eradication of Helicobacter pylori (H. pylori) in the infected area. The conventional clinical formulations of antibiotics have low specificity to H. pylori, which disrupts the normal balance of intestinal microbiomes. Therefore, oral drug delivery system with better stability at low pH as well as higher specificity to target H. pylori would provide more effective strategy to eradicate H. pylori and reduce the side effect of antibiotics. Based on the construction of UreI-mediated targeted drug delivery system developed by our group, in this work, using urea-modified UCCs-2 as targeting moiety to the UreI channel protein which is specifically expressed on H. pylori, pH-sensitive amoxicillin-loaded AMX-PLGA/UCCs-2 nanoparticles produced by UCCs-2 and PLGA for targeted treatment of H. pylori infection were established. The nanoparticles were prepared by double emulsion-solvent evaporation method. To achieve a promising drug delivery system with favorable pH-sensitive properties, we adopted an orthogonal design to obtain the optimal formulation. The results showed that the optimized AMX-PLGA/UCCs-2 nanoparticles were in a favorable pH sensitive manner and exhibited low cytotoxicity, higher specificity and better anti-H. pylori efficiency than amoxicillin and non-targeting AMX-PLGA/Cs nanoparticle both in vitro and in vivo, which can protect the antimicrobial drugs against acidic environment and deliver them to targeted eradicate H. pylori in the infected location. The cellular uptake mechanism showed that AMX-PLGA/UCCs-2 nanoparticles are an effective UreI-mediated targeted drug delivery system for anti-H. pylori treatment, which can also be used as promising nanocarriers for oral delivery of other therapeutic drugs to targeted treat H. pylori.