To alleviate the hemorrhagic side effect of thrombolysis therapy, a thrombus targeted drug delivery system based on the specific affinity of Annexin V to phosphatidylserine exposed on the membrane surface of activated platelet was developed. The amphiphilic and biodegradable biomaterial, polycaprolactone-block-poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-hydroxyethyl methacrylate) (PCL-b-PDMAEMA-b-PHEMA (PCDH)) triblock polymer, was synthesized via ring opening polymerization (ROP) and atom transfer radical polymerization (ATRP) to use as the nanocarriers of thrombolytic drug. In order to conjugate Annexin V to the polymer, PCDH was modified by succinic anhydride via ring-opening reaction to introduce the carboxyl group (PCDH-COOH). After preparation of PCDH/PCDH-COOH (9/1, m/m) mixed micelles, Annexin V was coupled with the micelles using carbodiimide chemistry. The blood clot lysis assay in vitro confirmed that lumbrokinase-loaded targeted micelles (LKTM) had stronger thrombolysis potency than free lumbrokinase (LK) and LK-loaded nontargeted micelles (LKM, P < 0.05). In vivo thrombolytic assay, multispectral, optoacoustic tomography (MSOT) was used to assess the target ability of LKTM. The results of MSOT images indicated the fluorescence intensity of the LKTM group located in the blood clot position were significantly stronger than the LKM group. A 5 mm of carotid artery containing blood clot was cut out 24 h later after administration to assess the degree of thrombolysis. The results of thrombolytic assay in vivo were consistent with the assay in vitro, which the differences between LK, LKM, and LKTM groups were both statistically significant. All the results of thrombolysis assays above proved that the capacity of thrombolysis in the LKTM group was optimal. It suggested that Annexin V-conjugated micelles will be a potential drug delivery system for targeted thrombolysis.