The development of recombinant insulin protein as a drug was a major step in the history of medicine. Today, the use of recombinant biological drugs is growing, which includes recombinant antibodies, a third of which is produced in Escherichia coli. Although these prokaryotic cells do not possess all the mechanisms for posttranslational modifications of proteins, E. coli has an oxidative environment for proteins in its periplasmic space where proteins can be directed for proper structuring. An important form of recombinant antibody, characterized by a very low molecular weight with preserved target molecule recognition properties, is the single-chain variable antibody fragment (scFv). The aim of this work is to investigate which factors result in higher yield and purity during scFv production in E. coli. Three methods of protein isolation from periplasm were used. Plasmid pOPE101 was used to transform E. coli pREP-DE3 strain with the scFv protein gene. The identity of the pOPE101-scFv expression vector was verified by restriction analysis, and high protein expression was achieved by overnight induction at 18°C. The scFv expression was confirmed by SDS-PAGE technique and Western blot analysis. We have shown that protein isolation from the periplasmic space is improved by vigorous stirring after cell resuspension and that double osmotic shock is the best technique for releasing soluble scFVs. It was observed that a large amount of scFv still remains in insoluble form. The obtained scFvs were also successfully isolated using the immobilized metal affinity chromatography (IMAC) and binding of the His tag, without loss during chromatography. To remove residual contaminants, a subsequent purification step based on size exclusion is recommended.