Cancer is a major cause of death in Thailand. Apart from surgery, chemotherapy, radiation, and hormonal therapy, cancer immunotherapy became the most promising treatment in the era. Immune checkpoint inhibitors (ICIs) act as immune stimulators to reactivate exhausted T cells to harness cancer cells. Programmed cell death protein 1 (PD-1) is the most potent immune checkpoint molecule, and PD-1-targeted ICIs have been approved by the U.S. FDA for cancer immunotherapy for over 30 indications. In Thailand, ICIs need to be imported for cancer treatment, and the cost of this drug is very expensive, and most cancer patients cannot access this kind of therapy. So, the development of this drug in our country is urgently needed to make it cheaper and increase the chance of patients to access the treatment. This study successfully developed a novel humanized anti-PD-1 monoclonal antibody (humanized anti-PD-1 CUSB1936) using mouse hybridoma technology and characterized its physicochemical and biological properties. The result showed that the humanized anti-PD-1 CUSB1936 could bind to PD-1 protein with high affinity in the sub-nanomolar range and neutralize the PD1/PD-L1 interaction with EC50 values 375.2 - 613.5 ng/mL. In addition, the anti-PD-1 CUSB1936 VH3+VL2 variant could induce T cell activation via IFN-gamma and IL-2 secretion in vitro better than the benchmark antibodies (Keytruda and Opdivo). Then, the anti-PD-1 CUSB1936 VH3+VL2 variant was transiently expressed using the Expi293 expression system. The purified anti-PD-1 CUSB1936 VH3+VL2 variant was then characterized and evaluated in vivo anti-tumor activity in MC38/hPD1 allograft mice model. The results exhibited that the anti-PD-1 CUSB1936 VH3+VL2 variant at 10 mg/kg could significantly inhibit tumor growth compared with IgG antibody control and showed similar antitumor efficacy compared with benchmark antibody. These findings prove that the anti-PD-1 CUSB1936 VH3+VL2 variant exhibits high anti-tumor efficiency and could be further developed as a novel PD-1 targeted ICI in Thailand.