Molecular imaging using single chain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form without any heterologous fusion is required for clinical applications. In this study, we expressed Thy1-scFv as a fusion protein with a N-terminal sequence including 3 hexa-histidines as purification tags together with a Trx-tag and a Stag for enhanced-solubility. Our strategy allowed to recover ∼35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the native protein with 97.7 ± 2.3 % purity without any tags. Finally, Thy1-scFv showed functionality towards its target on flow cytometry assays. In vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MBThy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) model (3.1 ± 1.2 a.u.) compared to control (0.4 ± 0.4 a.u.) suggesting potential for PDAC diagnosis. Overall, our strategy facilitates the expression and purification of a clinically translatable Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.