Pseudomonas aeruginosa (PA) is one of the most common multidrug-resistant pathogens found in clinics, often manifesting as biofilms. However, due to the emergence of superbugs in hospitals and the overuse of antibiotics, the prevention and treatment of PA infections have become increasingly challenging. Utilizing DNA nanostructures for packaging and delivering antibiotics presents an intervention strategy with significant potential. Nevertheless, construction of functional DNA nanostructures with multiple functionalities and enhanced stability in physiological settings remains challenging. In this study, the authors propose a magnesium-free assembly method that utilizes tobramycin (Tob) as a mediator to assemble DNA nanostructures, allowing for the functionalization of DNA nanostructures by combining DNA and antibiotics. Additionally, our study incorporates maleimide-modified DNA into the nanostructures to act as a targeting moiety specifically directed towards the pili of PA. The targeting ability of the constructed functional DNA nanostructure significantly improves the local concentration of Tob, thereby reducing the side effects of antibiotics. Our results demonstrate the successful construction of a maleimide-decorated Tob/DNA nanotube (NTTob-Mal) for the treatment of PA-infected lung inflammation. The stability and biocompatibility of NTTob-Mal are confirmed, highlighting its potential for clinical applications. Furthermore, its specificity in recognizing and adhering to PA has been validated. In vitro experiments have shown its efficacy in inhibiting PA biofilm formation, and in a murine model, NTTob-Mal has exhibited significant therapeutic effectiveness against PA-induced pneumonia. In summary, the proposed antibiotic drug-mediated DNA nanostructure assembly approach holds promise as a novel strategy for targeted treatment of PA infections.