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Our previous research verified that HSP (heat shock protein) 110 could enhance the anti-tumor effect of HPV16 E749-57 epitope. In this study, to optimize the immunotherapy of this vaccine type, we developed and evaluated the anti-tumor immunity of a nanoparticle vaccine format assembling with E749-57-HSP110 fusion expression plasmid and RGD-GGG-K18 polypeptide. The nanoparticle vaccine was self-assembled from positively charged RGD-GGG-K18 polypeptide and negatively charged fusion expression plasmid pIRES2-3× E7-HSP110-EGFP. The particle size, stability, expression of E749-57-HSP110 fusion protein and the target ability of nanoparticle were determined, respectively. Specific CTL responses were determined by E7 tetramer staining and cytotoxicity assay in TC-1 tumor-bearing mice (CD4/CD8 knockout). The preventive and therapeutic experiments of nanoparticle vaccine were investigated in TC-1 tumor-bearing mice. Results showed that the RGD-GGG-K18 polypeptide and pIRES2-3× E7-HSP110-EGFP plasmid self-assembled nanoparticles about 100 nanometers in diameter when the charge ratios of peptide/plasmid were 2. The nanoparticles effectively entered TC-1 cells directed by RGD target-peptide, and correctly expressed the E7-HSP110 fusion protein. The HSP110 effectively facilitated nanoparticles activating CD8+T cells than nanoparticles without HSP110, including the CD8+ T cell number and the IFN-γ level; in contrast, the CD4+T cells immune response remained indiscriminate among the mice groups. This nanoparticle formulation inhibited tumor growth and prolonged the survival duration in the prophylactic and therapeutic mouse models. Therefore, the RGD-based tumor-targeting nanoparticle expressing E749-57-HSP110 fusion protein can efficiently evoke anti-tumor activity and thus suggests it might be a favorable candidate for cervical cancer immunotherapy.