The secretory production of pharmaceutically or biotechnologically related recombinant proteins into the extracellular space of a bacterial expression system such as E. coli significantly simplifies their downstream processing and notably diminishes the production expenses, as well as time. Also, it causes a reduction in endotoxin contamination. Signal sequences play a significant role in proteins targeting translocation to the periplasmic compartment and release into the extracellular space during the secretion process. Natrecor is a recombinant drug used to treat the symptoms of Acutely Decompensated Heart Failure (HF). It is the only food and drug administration (FDA) approved drug to cure this disease. The enhancement of secretory expression of recombinant Natrecor protein using bacterial production machinery needs an appropriate signal sequence for high expression, prevention of misfolding, and simplified downstream purification processing. In the present research, to forecast the most appropriate signal sequence for high level secretory production of Natrecor in the prokaryotic host, 42 signal sequences were selected from bacterial sources and their essential features evaluated using different bioinformatics software. Therefore, their signal sequence probability and physicochemical characteristics were assessed using signalP software “version 4.1”, Portparam, and PROSO II severally. Following the evaluation of signal peptide-related parameters, ccmH, prsK, and fedA were selected in this study as the foremost options for the Natrecor expression in a secretory form (with D-scores 0.869, 0.832, and 0.815 severally). Therefore, they can be used potentially as proper nominees for impressive Natrecor production in the extracellular environment of E. coli. Later, the in silico cloning in pET-28a plasmid system was also carried out to ensure high expression of the best-selected signal peptide+ Natrecor in E. coli. It could be concluded that the computational software as a consensus method would be beneficial to forecast signal peptides with high reliability and has great popularity to choose and direct the modification of signal peptides for achieving a high-level secretory production of a given recombinant protein before starting experimental work.