We have developed a widely applicable method to construct epitope-peptide gene for epitope-vaccine strategy recently. In this study, we wanted to know whether the predefined spacers between epitopes on a recombinant epitope-peptide impacted the production of epitope-specific antibodies. The neutralizing epitope ELDKWA on the C-domain of HIV-1 gp41 was defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. We constructed three recombinant ELDKWA-epitope-peptides with different spacers between epitopes. The recombinant epitope-peptide GST-K8, GST-S8 and GST-R8 were bearing eight copies of ELDKWA-epitope with amino acid spacer GS, GSGGGGS and RS, respectively. GST-K8 and GST-S8 could induce high titer of ELDKWA-epitope-specific antibodies, much better than GST-R8. Besides, both antibodies could recognize the recombinant soluble gp41 and the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that the spacer GSGGGGS and GS were feasible in constructing a recombinant epitope-vaccine.