Atherosclerosis is a leading cause of death globally. Targeted imaging and therapeutics are desirable for the detection and treatment of the disease. In this study, we developed trifunctional Simian virus 40 (SV40)-based nanoparticles for in vivo targeting and imaging of atherosclerotic plaques. These novel trifunctional SV40-based nanoparticles encapsulate near-infrared quantum dots and bear a targeting element and a drug component. Using trifunctional SV40-based nanoparticles, we were able to noninvasively fluorescently image atherosclerotic plaques in live intact ApoE(-/-) mice. Near-infrared quantum dots encapsulated in the SV40 virus-like particles showed prominent optical properties for in vivo imaging. When different targeting peptides for vascular cell adhesion molecule-1, macrophages, and fibrin were used, early, developmental, and late stages of atherosclerosis could be targeted and imaged in live intact ApoE(-/-) mice, respectively. Targeted SV40 virus-like particles also delivered an increased concentration of the anticoagulant drug Hirulog to atherosclerosis plaques. Our study provides novel SV40-based nanoparticles with multivalency and multifunctionality suitable for in vivo imaging, molecular targeting, and drug delivery in atherosclerosis.