The use of nanoparticles (NPs) has been of great interest in the field of vaccinology for the generation and development of nanocarriers for new oral vaccines. Zein NPs have become a promising option since protein NPs show several advantages, such as biocompatibility and a longer antigen release. To use these NPs as an oral vaccine model, they can be conjugated with a molecule that improves their recognition by the intestinal dendritic cells. Folic acid has been used due its high affinity for dendritic cells, making it an ideal molecule for conjugation with NPs. In this work, a relatively simple and highly reproducible method was established to generate zein-based nanocarriers: pure zein NPs and zein NPs with covalently linked folate. Two different strategies for covalent bond formation between the zein NPs and NHS–folate were evaluated, one for the formation of zein NPs with folate on their surface (ZN-FA NPs) and another one for zein NPs with folate both on their surface and inside of them (ZN(FA) NPs). The obtained NPs were characterized in terms of shape, size, polydispersity, and zeta potential using dynamic light scattering (DLS) analysis and visualized via transmission electron microscopy (TEM). The synthesized ZN-FA NPs and ZN(FA) NPs showed a spherical shape with low polydispersity and an average particle size of 229.88.42 ± 59.52 nm and 293.70 ± 69.19 nm, respectively, according to the TEM analysis, while the DLS analysis showed a hydrodynamic radius of 366.42 ± 39.84 nm and 455.77 ± 91.55 nm, respectively. Fourier-transform Infrared spectroscopy (FTIR) confirmed the successful conjugation of folate to the NPs, while UV-Vis spectrophotometry determined the concentration of folate in the NPs and a protein adsorption efficiency of 45.44% and 41.00% for ZN-FA NPs and ZN(FA) NPs, respectively. Furthermore, cytotoxicity assays in the DC2.4 dendritic cell line demonstrated that the NPs are not cytotoxic.