The activation of Toll-like receptors (TLR) by natural or synthetic ligands results in cytokine secretion and increased phagocytosis by macrophages and cytolytic activity by natural killer (NK) cells. So,wedeveloped a stable, efficient, and nontoxic cationic nanoemulsion (CNE) suitable for TLR ligand oligonucleotide (ssRNA) delivery. The nanoemulsion is based on squalene, cationic lipid 1,2-dioleoyl-sn-glycero-3-trimethylammoniumpropane (DOTAP), helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), tween 80, and poloxamer 188. Factorial design was used to investigate the influence of of tween 80 and poloxamer 188 on particle size, single strand RNA (ssRNA) binding %, Interferon (INF α induction). Results showed that by increasing the poloxamer 188 and/or tween 80, the particle size decreased.The influence of tween 80 and poloxamer 188 concentrations on the particle surface on ssRNA binding efficiency is evident. Although most of the formulations showed a high binding efficiency of ssRNA, only two formulations produced high amount of INF α. Interestingly that in both formulations either poloxamer 188 is present (F3) or tween 80 is present (F4) but where both together in one formulation, the INF α production decreased relatively. In conclusion, cationic nanoemulsion could be a promising drug delivery system for nucleic acids (DNA/RNA) in cancer and viral immunotherapy.