To improve its antitumor effect, we used human leukocyte antigen -A2 (HLA-A2)-positive human dendritic cell (DC)-derived DEXs (DC-derived exosomes) to support NY-ESO-1 antigen and polyI:C, with the aim of increasing the proliferation of specific cytotoxic T lymphocytes (CTL) in transgenic mice. Mature dendritic cells derived from peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adults with positive HLA-2A. Using centrifuge and membrane ultrafiltration, EXO (exosomes) were extracted from the supernatant of DCs secretions. Transgenic C57 mice were immunized with human-derived tumor testis antigen NY-ESO-1/EXO, with or without polyI:C. Mice were sacrificed four weeks after immunization, and spleen cells were isolated and tested for function. The experiments included antigen-specific CTL proliferation, as tested by dimerization and antitumor effects for K562 cells as well as melanoma, tested at different ratios of effected cells:target cells (0:1, 10:1, 50:1, and 100:1). Dimerization experiments indicated that the effect of DEX/TSA (tumor specific antigens) + PolyI:C was 2.36 ± 1.10% and the control was 0.38 ± 0.31%, while the effect of DEX/TSA was 1.97 ± 0.63% and the control was 0.36 ± 0.07%. Antitumor effects by DEX/TSA: PolyI:C for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 11.14 ± 1.36%, 14.17 ± 0.62%, 15.71 ± 2.48%, and 24.31 ± 2.91%, respectively, for K562 cells. The antitumor effects for DEX/TSA for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 12.23 ± 2.25%, 13.10 ± 1.57%, 15.27 ± 2.93%, and 19.87 ± 2.72%, respectively, for K562 cells. With ratios of 10:1 and 100:1, the antitumor effects of DEX/TSA + PolyI:C were better than for the DEX/TSA group (P < 0.05). However, higher ratios of effecter cells to target cells increased, and there were no significant improvements in antitumor effect for control cells. Combining PolyI:C with DEX/TSA derived from healthy human blood positive for HLA-A2 is a promising strategy for developing new subcellular antitumor vaccination.