The purpose of this study was to test the feasibility of using a novel biodegradable microsphere as a contrast agent/gene vector in ultrasound imaging-based vascular gene delivery. We first encapsulated green fluoresent protein (GFP) gene into the microspheres and validated the echogenic property of GFP-plasmid/microspheres with intramuscular injection. Then, the GFP-plasmid/microspheres were transferred into the arteries of pigs via a catheter-based delivery procedure under ultrasound imaging. Strong echogenic signal reflected from the microspheres indicated the destination of genes delivered, whereas functional gene expression was confirmed by immunohistochemical examination. Microspheres can be feasible contrast agents/gene vectors in ultrasound imaging-based vascular gene delivery in vivo.