The aim of the present study is to establish a bacterial clone capable of secreting an integrin αvβ3 targeting probe with bioluminescent and fluorescent activities, and to verify its specific targeting and optical activities using molecular imaging. A bacterial vector expressing a fusion of secretory Gaussia luciferase (sGluc), mCherry, and RGD (sGluc-mCherry-RGDX3; GCR), and a control vector expressing a fusion of secretory Gaussia luciferase and mCherry (sGluc-mCherry; GC) were constructed. The GCR and GC proteins were expressed in E. coli and secreted into the growth medium, which showed an approximately 10-fold higher luciferase activity than the bacterial lysate. Successful purification of GCR and GC was achieved using the 6X His-tag method. The GCR protein bound with higher affinity to U87MG cells than CHO cells in confocal microscopy and IVIS imaging, and also showed a high affinity for integrin αvβ3 expressing tumor xenografts in an in vivo animal model. An E. coli clone was established to secrete an integrin αvβ3 targeting imaging probe with bioluminescent and fluorescent activities. The probe was produced feasibly and at low cost, and has shown to be useful for the assessment of angiogenesis in vitro and in vivo.