Mesothelin is an epithelial marker highly expressed at the cell surface of cancer cells from diverse origins， including ovarian and pancreatic adenocarcinomas and mesotheliomas. Previously， we identified and characterized an antimesothelin nanobody (NbG3a) for in vitro diagnostic applications. The main goal of this research was to establish the potential of NbG3a as a molecular imaging agent. Site-specific biotinylated NbG3a (bNbG3a) was bound to streptavidin-conjugated reagents for in vitro and in vivo assays. Initially， we performed microscale thermophoresis to determine the binding affinity between bNbG3a and human ( K = 46 ± 8 nM) or mouse ( K = 4.8 ± 0.4 nM) mesothelin protein. The human and mouse cross-reactivity was confirmed by in vivo optical imaging using bNbG3a bound to fluorescent streptavidin. We also localized the binding site of nNbG3a on human mesothelin using overlapping peptide scan. NbG3a recognized an epitope within residues 21-65 of the mature membrane bound form of human mesothelin， which is part of the N-terminal region of mesothelin that is important for interactions between mesothelin on peritoneal cells and CA125 on tumor cells. Next， the bNbG3a in vivo half-life after intravenous injection in healthy mice was estimated by ELISA assay to be 5.3 ± 1.3 min. In tumor-bearing animals， fluorescent bNbG3a accumulated in a subcutaneous ovarian xenograft (A1847) and in two syngeneic， orthotopic ovarian tumors (intraovary and intraperitoneal ID8) within an hour of intravenous injection that peaked by 4 h and persisted up to 48 h. MRI analysis of bNbG3a-targeted streptavidin-labeled iron oxides showed that the MRI signal intensity decreased 1 h after injection for a subcutaneous xenograft model of ovarian cancer for bNbG3a-labeled iron oxides compared to unlabeled iron oxides. The signal intensity differences continued up to the final time point at 24 h post injection. Finally， in vivo immunofluorescence 24 or 48 h after bNbG3a intravenous injection showed bNbG3a diffuse distribution of both xenograft and syngeneic ovarian tumors， with local areas of high concentration throughout A1847 human tumor. The data support the use of NbG3a for continued preclinical development and translation to human applications for cancers that overexpress mesothelin.