CD27, a costimulatory molecule of the TNF receptor superfamily expressed on T lymphocytes, plays a critical role in regulating T cell survival, differentiation, and effector function. Upon binding its ligand, CD70, CD27 signaling enhances T cell proliferation and differentiation into effector and memory T cells. This agonistic activity positions CD27 as a promising target for immunomodulatory cancer therapy. This study investigates the development of therapeutic agents targeting the CD27 for cancer immunotherapy. We employed a recombinant human CD27 extracellular domain to immunize an alpaca (Vicugna pacos), generating antigen-specific single-domain antibodies (nanobodies). Through biopanning using the immunized phage display library, we identified the anti-CD27 cpNb4 clone exhibiting high binding affinity for recombinant human CD27 and specific binding to cell-surface CD27 expressed on CHO-K1 cells. These findings establish cpNb4 as a promising candidate for further investigation, potentially paving the way for its integration into combination immunomodulatory cancer therapy regimens.