Objective To present atherosclerotic plaques via fluorescence imaging by designing and constructing a probe Cy5.5-anti-OPN-DMSA-MNPs (OPN: osteopontin, DMSA-MNPs: meso-2,3-dimercaptosuccinic acid-Fe3O4 magnetic nanoparticles). Methods OPN antibody and Cy5.5 dye were conjugated to DMSA-MNPs by amide reaction between carboxylic group and amine group. The cytotoxicity of probes for mouse macrophages was evaluated by MTT and TUNEL assays. ApoE?/? mice were fed with high fat diet for 20 weeks to build atherosclerosis models, and then were given an injection of the probes (5mg Fe/kg) via tail vein. Fluorescence imaging was performed in vivo by IVIS Kinetic System 24h later. Then, the carotid arteries were collected for frozen section examination by confocal fluorescent microscopy. Results The MTT results showed that there was no significant discrepancy in cell viability between the macrophages treated by 5, 10, 15, 20, 25 and 30mg/L probe respectively and the control cells (A490nm: 1.10±0.03, 1.05±0.03, 1.03±0.02, 0.96±0.02, 0.96±0.03, 0.93±0.03 vs 1.11±0.05, P＞0.05). The TUNEL indicated that no significant difference was found in the number of positive nuclei in the treated cells (10, 20, 25 and 30mg/L probe) and control cells [(21.2±1.5)%, (21.8±1.1)%, (21.5±1.2)%, (22.3±1.2)% vs (20.5±1.0)%, P＞0.05]. These results exhibited that the probe had no influence on cell survival at above-used concentrations. After the probes were injected into the atherosclerosis models, the fluorescence imaging clearly displayed the signal of atherosclerotic plaques in carotid artery, and confocal fluorescent microscopy showed that the probes were mainly located in the plaque which was further verified by HE staining. Conclusion Our self-made probes have barely no cytotoxicity on cells within the concentrations we used, and are effective in detection of atherosclerotic plaques in carotid artery in vivo.