Hypoparathyroidism is a rare endocrine disease which is characterized by the deficiency of serum calcium levels. RhPTH is prescribed as a therapy for the management of refractory hypoparathyroidism. The aim of this study is to investigate 32 signal peptides of gram-negative bacterial origin and evaluate their potential for efficient secretion of recombinant human PTH (1–84)In E.coli to obtain higher expression of recombinant PTH in bacterial systems by using this fusion partner. SignalP and ProtParam servers were employed to predict the presence and location of signal peptide cleavage sites in protein sequence and computation of various physical and chemical parameters of protein respectively. Also, SOLpro server was applied for prediction of the protein solubility. Then ProtComp and SecretomeP online servers were employed to determine protein location. The evaluations showed that theoretically two signal peptides Lipopolysaccharide export system protein LptA (lptA) and Periplasmic pH-dependent serine endoprotease DegQ (degQ) are the most appropriate signal peptides examined. Due to the lack of post-translational modification in PTH, its periplasmic expression has preferences. Based on the results of this study, using bioinformatics and reliable servers signal peptides with appropriate secretory potential can be obtained which lead to the highest expression level.