Recent research has focused on immunotherapy, particularly with regard to cancer treatment. Programmed death-1 and programmed death ligand 1 (PD-1/PD-L1) pathway blockade is a central topic of the promising immunotherapy field. In veterinary medicine, observations of the PD-1/PD-L1 pathway, including the relationship between immune cells and diseases, have increased. In this study, monoclonal antibodies specific to canine PD-1 and PD-L1 were developed, and the antibodies against PD-1 and PD-L1 bind to PD-1 and PD-L1 overexpressing cells, respectively. Additionally, each antibody interfered with the interaction between PD-1 and PD-L1. The expression of PD-1 and PD-L1 was detected on activated T cells from canine peripheral blood mononuclear cells (PBMC), and, remarkably, was the first recorded instance of PD-L1 expression on canine immature dendritic cells. Production of IFN-γ by activated T cells increased significantly when incubated with anti-PD-1 antibody alone and with both anti-PD-1 and anti-PD-L1 antibodies, revealing the functional effects of the antibodies. The antibodies will be useful for research on immune systems and may be the first passive immunotherapy approach in canine cancer patients.