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 fluorescent 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. A 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.