Silica-based nanoparticles are promising carriers for gene delivery applications. To gain insights into the effect of particle size on gene transfection efficiency, amine-modified monodisperse Stöber spheres (NH₂-SS) with diameters of 125, 230, 330, 440, and 570 nm were synthesized. The in vitro transfection efficiencies of NH₂-SS for delivering plasmid DNA encoding green fluorescent protein (GFP) (pcDNA3-EGFP, abbreviated as pcDNA, 6.1 kbp) were studied in HEK293T cells. NH₂-SS with a diameter of 330 nm (NH₂-SS330) showed the highest GFP transfection level compared to NH₂-SS particles with other sizes. The transfection efficiency was found as a compromise between the binding capacity and cellular uptake performance of NH₂-SS330 and pcDNA conjugates. NH₂-SS330 also demonstrated the highest transfection efficiency for plasmid DNA (pDNA) with a bigger size of 8.9 kbp. To our knowledge, this study is the first to demonstrate the significance of particle size for gene transfection efficiency in silica-based gene delivery systems. Our findings are crucial to the rational design of synthetic vectors for gene therapy.