The secretory production of heterologous proteins in E. coli has revolutionized biotechnology. Efficient periplasmic production of foreign proteins in E. coli often requires a signal peptide to direct proteins to the periplasm. However, the presence of attached signal peptide does not guarantee periplasmic expression of target proteins. Overproduction of auxiliary proteins, such as chaperones can be a useful approach to enhance protein export. In the current study, three chaperone plasmid sets, including GroEL-GroES (GroELS), Dnak-Dnaj-GrpE (DnaKJE), and trigger factor (TF), were coexpressed in E. coli BL21 (DE3) in a pairwise manner with two pET22-b vectors carrying the recombinant hirudin-PA (Hir) gene and different signal sequences alkaline phosphatase (PhoA) and L-asparaginase II (L-ASP). Overexpression of cytoplasmic combinations of molecular chaperones containing GroELS and DnaKJE with PhoAHir increased the secretory production of PhoAHir by 2.6fold (p < 0.05) and 3.5fold (p < 0.01) compared with their controls, respectively. By contrast, secretory production of PhoAHir significantly reduced in the presence of overexpressed TF (p = 0.02). Further, periplasmic expression of L-ASP was significantly increased only in the presence of DnaKJE (p = 0.04). These findings suggest that using molecular chaperones can be helpful for improving periplasmic expression of Hir. However, tagged signal peptides may affect the physicochemical properties and secondary and tertiary structures of mature Hir, which may alter their interactions with chaperones. Hence, using overexpressed chaperones has various effects on secretory production of PhoAHir and L-ASPHir.