T-20 is a human immunodeficiency virus (HIV) fusion inhibitor, consisting of 36 amino acids, which blocks the fusion between the membrane of the host immune cell T-lymphocyte and the HIV-1 virus. We investigated various fusion and tandem-repeated peptide expression systems in Escherichia coli for the cost-efficient production of T-20 peptide. A gene module (referred to as EJT20) was designed based on the codon preference of E. coli, and various tandem repeats (EJT20n, where n represents the number of gene modules) were constructed to enhance the expression level of T-20 precursor. Among the different expression systems constructed, pKSI-EJT203 containing trimeric EJT20 fused to KSI (ketosteroid isomerase) gene as a fusion partner was expressed up to 60% of the total cell proteins under the control of T7 lac promoter.