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Objective: To evaluate the effect of anti-OX40 and anti-AFP antibodies conjugated onto poly(DL-lactide-co-glycolide)-nanoparticles (PLGA-NPs) on the cytotoxic activity of AFP158-166; -specific cytotoxic T lymphocyte (CTL) against hepatocellular carcinoma cells in vitro., Methods: PLGA-NPs were prepared by oil-in-water single emulsion solvent evaporation method and covalently conjugated with anti-OX40 and anti-AFP monoclonal antibodies. Scanning electron microscopy (SEM) was utilized for the characterization of the surface morphology and estimation of the size of the PLGA-NPs. The mean diameter and zeta potential of the nanoparticles were measured by dynamic light scattering (DLS) performed in a Zetasiser Nano Series ZEN3600. Antibody conjugation efficiency was determined using bicinchoninic acid (BCA) protein assay. Dendritic cells (DCs) were induced from human peripheral blood mononuclear cells (PBMCs) in the presence of GM-CSF and IL-4, and loaded with AFP158-166; peptide to generate AFP-specific CTL (CTL/AFP158-166;). WST-1, ELISA and lactate dehydrogenase (LDH) methods were respectively used to examine the effects of the anti-OX40/anti-AFP-NPs on CTL/AFP158-166; proliferation, IL-2 and IFN-γ production, and cytotoxicity against the tumor cells., Results: The obtained nanoparticles were found to be of regular spherical shape and the smooth surface with an average diameter of (300±42) nm and a negative zeta potential of -(25.12±5.34) mV. Approximately 100 μg antibodies were conjugated to every milligram of the nanoparticles with a conjugation efficiency of about 25% as estimated by BCA protein assay. Proliferation and activation analysis revealed that anti-OX40/anti-AFP mAb-NPs significantly induced CTL proliferation and the secretion of IL-2 and IFN-γ. The cytotoxicity assay showed that anti-OX40/anti-AFP-NPs markedly enhanced CTL/AFP158-166; specific killing on HepG2 cells but had no obvious effect on SMMC-7721 cells., Conclusion: Anti-OX40 mAb and anti-AFP mAb conjugated to PLGA-NPs could stimulate CTL/AFP158-166; cell proliferation and cytokine production as well as enhancing their specific killing on AFP-positive hepatocellular carcinoma cells.