Purpose: Gene therapy is an important therapeutic strategy for cancer. Nanoparticles are used for noninvasive gene delivery, which has great potential in tumor therapy. However, it is a challenge to construct a targeted gene delivery vector with high gene delivery efficiency, good biocompatibility, and multiple functions. Method: Herein, we designed magnetic mesoporous silica nanoparticle loading microbubbles (M-MSN@MBs) for ultrasound-mediated imaging and gene transfection. The plasmid DNA (pDNA) was encapsulated into the pores of M-MSNs. Also, the pDNA-carrying M-MSNs were loaded in the lipid microbubbles. Results: The gene vector presented good biocompatibility, DNA binding stability, ultrasound imaging performance, and magnetic responsiveness. The polyethyleneimine (PEI)-modified M-MSNs effectively protected the loaded pDNA from enzyme degradation. The cytotoxicity of M-MSNs was significantly reduced via encapsulating in lipid microbubbles. Upon the magnetic field, M-MSN@MBs were attracted to the tumor area. Then, ultrasound-targeted microbubble destruction (UTMD) not only released loaded M-MSNs but also facilitated M-MSNs delivery to tumor tissue by opening blood-tumor barrier and increasing the cytomembrane permeability, and ultimately improved the pDNA delivery efficiency. Conclusion: Our findings suggested that the developed ultrasound-responsive gene delivery system was a promising platform for gene therapy, which could noninvasively enhance tumor gene transfection.