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Background: Immune checkpoints are molecules that act as regulators of immune system pathways. However, some tumor cells can express the ligands of immune checkpoints to escape from antitumor immune responses. Some agents, such as antibodies, can inhibit these checkpoints that prevent the immune system from targeting and killing cancer cells. The aim of this study was to express a novel bispecific tandem scFv in periplasmic space of Escherichia coli for simultaneous targeting of two immune checkpoints, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Materials and Methods: The bispecific tandem scFv was constructed based on the variable regions gene of anti-PD1 and anti-CTLA-4 antibodies. The optimum codon for expression in E. coli was chemically synthesized and subcloned in periplasmic expression plasmid. After transformation, the effect of cultivation conditions on periplasmic expression of the protein in E. coli BL21(DE3) was evaluated. Then, the bispecific tandem scFv was purified and its binding ability to cells expressing PD-1 and CTLA-4 was evaluated. Results: Expression of tandem scFv with a molecular weight of 55 kDa was verified by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting analysis. The best condition for soluble periplasmic expression was obtained to be incubation with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 23°C. The protein was successfully purified using affinity chromatography with a final yield of 4.5 mg/L. Binding analysis confirmed the bioactivity of purified the tandem scFv. Conclusion: This bispecific tandem scFv could be a potential candidate to cancer immunotherapy, although more biological activity assessments are still required to be carried out.