AbstractMethicillin‐resistant Staphylococcus aureus (MRSA) biofilm‐associated bacterial keratitis is highly intractable, with strong resistance to β‐lactam antibiotics. Inhibiting the MRSA resistance gene mecR1 to downregulate penicillin‐binding protein PBP2a has been implicated in the sensitization of β‐lactam antibiotics to MRSA. However, oligonucleotide gene regulators struggle to penetrate dense biofilms, let alone achieve efficient gene regulation inside bacteria cells. Herein, an eye‐drop system capable of penetrating biofilms and targeting bacteria for chemo‐gene therapy in MRSA‐caused bacterial keratitis is developed. This system employed rolling circle amplification to prepare DNA nanoflowers (DNFs) encoding MRSA‐specific aptamers and mecR1 deoxyribozymes (DNAzymes). Subsequently, β‐lactam antibiotic ampicillin (Amp) and zinc oxide (ZnO) nanoparticles are sequentially loaded into the DNFs (ZnO/Amp@DNFs). Upon application, ZnO on the surface of the nanosystem disrupts the dense structure of biofilm and fully exposes free bacteria. Later, bearing encoded aptamer, the nanoflower system is intensively endocytosed by bacteria, and releases DNAzyme under acidic conditions to cleave the mecR1 gene for PBP2a down‐regulation, and ampicillin for efficient MRSA elimination. In vivo tests showed that the system effectively cleared bacterial and biofilm in the cornea, suppressed proinflammatory cytokines interleukin 1β （IL‐1β） and tumor neocrosis factor‐alpha （TNF‐α）, and is safe for corneal epithelial cells. Overall, this design offers a promising approach for treating MRSA‐induced keratitis.