Abstract: Lipid nanoparticles (LNP) are highly promising gene delivery platform due to their high safety, good stability, and excellent nucleic acid encapsulation efficiency. However, current clinical gene therapy primarily relies on invasive administration routes. The development of compliant, non-invasive delivery routes, such as oral administration, is crucial. Nevertheless, creating gastric acid-resistant oral gene delivery vectors faces a critical bottleneck that nucleic acid therapeutics are highly susceptible to degradation in the harsh acidic environment (pH 1.5-4.5) of the gastrointestinal tract. To address this, we constructed a cholesterol-enriched hybrid system (RLSC/OMVC) based on an amphiphilic cationic lipopeptide (RLS) and Escherichia coli outer membrane vesicles (OMV). This system demonstrates efficient gene delivery capability under both neutral and acidic conditions, along with excellent in vitro biocompatibility. The study characterized the physicochemical properties of different formulations (RLS, RLSC, OMV, and OMVC). In vitro cell transfection experiments showed that the gene delivery efficiency of RLSC/OMVC under acidic condition (pH 4.5) was significantly increased by nearly 3-fold compared with Lipofectamine 2000. Furthermore, transmission electron microscopy observation, particle size/zeta potential analysis, and gel electrophoresis experiments revealed the mechanism by which OMV and cholesterol synergistically protect plasmid DNA from acidic degradation in conjunction with lipid peptide. The developed RLSC/OMVC hybrid system boasts integrated advantages of exceptional acid resistance, high-efficiency gene delivery, and good biocompatibility. This work provides an innovative strategy for oral gene therapy.