<p>Self-amplifying mRNA (SAM) shows promise for vaccines and gene therapy because of its self-replicating ability. However, current studies lack sufficient information for systematic parameter optimization and differentiation from conventional non-replicating mRNA (NRM). Therefore, the transfection efficiency of NRM and SAM platforms was evaluated by comparing delivery vectors and optimizing parameters for the SAM protocol. SAM and NRM showed similar transfection preferences, but their efficiencies differed. Optimized SAM transfection parameters were then established, including dose and incubation time. In this study, an in vitro multi-parameter delivery system for SAM was constructed, providing valuable insights into SAM transfection and its distinction from regular mRNA. This study contributes an experimental basis for the rational screening of nucleic acid drug carriers and the establishment of SAM multi-parameter evaluation criteria, and also lays an important foundation for optimizing low-dose immunization strategies and their clinical application translation.</p> Graphical Abstract <p></p>