Previous studies indicate that immunostimulatory DNA-based injectable hydrogels harboring unmethylated cytosine-phosphate-guanine (CpG) dinucleotides meet the requirements of an effective antigen delivery system, including safety, biodegradability, ease of administration, and stimulation of the innate immune system. However, rapid release of the model antigen ovalbumin (OVA) from the hydrogel limits its potential. Here, the aim is to achieve sustained OVA release from a DNA hydrogel through cationization of the antigen. Ethylenediamine (ED)-conjugated cationized OVA (ED-OVA), but not OVA, forms a complex with hexapod-like structured DNA, a component of the DNA hydrogel. The release of ED-OVA from the hydrogel is significantly slower than that of OVA. ED-OVA mixed with CpG DNA hydrogel efficiently binds to mouse dendritic DC2.4 cells and results in high antigen presentation. Intratumoral injections of ED-OVA/CpG DNA hydrogel significantly delays tumor growth of OVA-expressing EG7-OVA cells in mice. Then, a cationic OVA peptide antigen (R8-L2-pepI) consisting of an OVA MHC class I epitope, octaarginine, and a linker is designed. Intratumoral injections of R8-L2-pepI/CpG DNA hydrogel eradicate tumors in five out of six mice. Thus, it is concluded that a vaccine consisting of immunostimulatory CpG DNA hydrogel and cationized antigens can be effective for cancer immunotherapy.