Melanoma is among the few cancers with rising incidence. Currently there is no effective treatment for metastatic disease, but improved detection of melanoma has the potential to benefit the management of patients with early disease. Radioimmunodection by imaging with single-chain Fv (scFv) antibody fragments is one such emerging diagnostic method. However, the amount of scFv that can be produced at a scale suitable for use in patients is limiting. We have previously shown that the bacterial expression of a scFv derived from a monoclonal antibody (MAb) specific for melanoma-associated proteoglycan can be increased by light chain shuffling. In this report we show that a further increase in expression yield can be obtained by reversing the usual V(H)-V(L) orientation of scFvs to V(L)-V(H). Such seemingly minor changes have previously been reported to have unexpected effects on the in vitro and in vivo binding properties of recombinant antibodies. Our results show that reversal of the V domain orientation of the scFv improves expression by 150% without an adverse effect on melanoma binding in vitro and tumor targeting in vivo. Therefore, our results show that alteration of V domain orientation can improve the production yield of clinically useful antibody fragments. When used in combination with other antibody engineering approaches for increased antibody production changing the domain orientation is a simple strategy to achieve significant improvements in the production of scFvs for tumor radioimmunodetection for patient studies.