To develop a new kind of non-viral inorganic gene vector, embedded dual-mesoporous silica spheres (EDMSNs) was modified with polyethyleneimine (PEI) or 3-aminopropyltriethoxysilane (APTMS). Then gene loading and transfection ability of the obtained EDMSNs-PEI and EDMSNs-NH₂ was tested by choosing the pCMV-EGFP-N1 plasmids (pDNA) as a gene model. Particle morphologies, particle sizes, zeta potentials, and pore structure parameters of EDMSNs-NH₂ and EDMSNs-PEI were characterized by transmission electron microscopy, dynamic light scattering and nitrogen adsorption-desorption. Both EDMSNs-NH₂ and EDMSNs-PEI display obvious dual-mesoporous structure and well-defined spherical morphology. Average dynamic sizes of EDMSNs-NH₂ and EDMSNs-PEI are 343.2 and 338.9 nm, while zeta potentials of EDMSNs-NH₂ and EDMSNs-PEI are +18 and +43 mV, respectively. The results of gel electrophoresis, CCK-8 assay, and fluorescence microscope show that the pDNA loading amount of EDMSNs-PEI is 8%, much higher than that (1%) of EDMSNs-NH₂. In addition, EDMSNs-PEI exhibits lower cytotoxicity than that of pure PEI polymers and lipofectamine2000. When the mass ratio of EDMSNs- PEI/pDNA is 33 : 1, the transfection rate of the 293T cells by EDMSNs-PEI/pDNA reaches a maximum at 72 h. In conclusion, EDMSNs-PEI, compared with EDMSNs-NH2, has a higher positive potential and pDNA loading capacity, which can be used as a promising non-viral inorganic gene vector for the treatment of diseases such as malignant glioma.