Cancer remains a significant global health issue, and conventional treatments often fail to provide satisfactory outcomes for many patients. Immunotherapy, particularly the inhibition of immune checkpoints, has emerged as a promising approach to prevent tumor recurrence and metastasis. Cytotoxic T lymphocyte-associated protein-4 (CTLA-4) is a crucial immune molecule involved in cell cycle modulation, T cell proliferation, and cytokine production. Inhibiting overexpression of immune checkpoints like CTLA-4 has been confirmed as an effective strategy in cancer therapy. Nanobodies, derived from the variable domain of heavy antibody chains, are small protein fragments with unique properties that make them ideal tools for imaging and treating diseases. They have low molecular weight, high stability, and specific binding to targets, making them effective in overcoming the limitations of monoclonal antibody-based immunotherapy. This article reviews the cellular and molecular functions of CTLA-4, the structure and mechanisms of nanobodies, and their delivery methods. It highlights the advantages and challenges of using nanobodies in cancer immunotherapy, emphasizing the potential of anti-CTLA-4 nanobodies. The article also discusses the development of nanobody-based strategies to inhibit CTLA-4 checkpoint, which could have promising results in treating both hematological and solid tumors. However, several open questions remain, including the success of nanobody-based drugs in cancer treatment and diagnosis, the clinical efficacy of anti-CTLA-4 nanobodies, and solutions to overcome the limitations of producing modified nanobodies with Fc-receptors.Cancer remains a significant global health issue, and conventional treatments often fail to provide satisfactory outcomes for many patients. Immunotherapy, particularly the inhibition of immune checkpoints, has emerged as a promising approach to prevent tumor recurrence and metastasis. Cytotoxic T lymphocyte-associated protein-4 (CTLA-4) is a crucial immune molecule involved in cell cycle modulation, T cell proliferation, and cytokine production. Inhibiting overexpression of immune checkpoints like CTLA-4 has been confirmed as an effective strategy in cancer therapy. Nanobodies, derived from the variable domain of heavy antibody chains, are small protein fragments with unique properties that make them ideal tools for imaging and treating diseases. They have low molecular weight, high stability, and specific binding to targets, making them effective in overcoming the limitations of monoclonal antibody-based immunotherapy. This article reviews the cellular and molecular functions of CTLA-4, the structure and mechanisms of nanobodies, and their delivery methods. It highlights the advantages and challenges of using nanobodies in cancer immunotherapy, emphasizing the potential of anti-CTLA-4 nanobodies. The article also discusses the development of nanobody-based strategies to inhibit CTLA-4 checkpoint, which could have promising results in treating both hematological and solid tumors. However, several open questions remain, including the success of nanobody-based drugs in cancer treatment and diagnosis, the clinical efficacy of anti-CTLA-4 nanobodies, and solutions to overcome the limitations of producing modified nanobodies with Fc-receptors.