Signal peptides (SPs) are essential tools to keep folding and function of recombinant proteins which are folded by disulfide bonds. Human serum albumin (HSA) is a protein with medical application which is folded by 17 disulfide bonds. Therefore, the production of this protein as recombinant protein needs appropriate SP. This study was performed to identify the best SP to express HSA in Escherichia coli. At first, 42 signal sequences (SSs) were collected from the database, then SP probability and SP regions were predicted by SignalP (version 4). Physico-chemical features of SPs were also evaluated by Portparam, whereas signal solubility was assessed by SOLpro. Finally secretion sorting of SPs were investigated by PRED-TAT server. The results of this study revealed that, among 42 signal sequences, FlgI, DsbG, OmpC and MepA (respectively) are theoretically the best SPs to express HSA in E. coli.