Cancer immunotherapy to harness the patient own immune system fighting against cancer is a breakthrough of cancer treatment. T-cell therapy has been successful for treatment of leukemia and melanoma, driving immunotherapy to possibly be front line of cancer treatment. However, the rate of its successfulness in vivo has been reported to be dramatically affected by immune escape mechanism, a major of which is through the interaction between PD-1 and PDL1 that results in induction of T cell exhaustion and promotion of cancer evasion from the body immune surveillance. Interruption PD-1/PD-L1 interaction is a crucial approach to enhance T-cell immunotherapy in vivo. This study, therefore, aimed to identify single chain variable fragment (ScFv) specific to PD-L1 for inhibition of PD-1/PD-L1 interaction. Initially, anti-PD-L1 ScFvs were screened from human ScFv-phage display ‘YAMO’ library. After testing protein expression, 11 unique ScFv clones were identified by DNA fingerprint using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Binding specificity of ScFv proteins to PD-L1 on KKU-213 cholangiocarcinoma cells was examined by flow cytometry. Top 5 ScFv clones were selected to analyze by DNA sequencing. The deduced amino acid sequences were obtained and predicted by using IMGT/V-QUEST software. The deduced amino acid sequences were aligned and predicted for putative ScFv structures by using Clustal Omega software and SWISS-MODEL webserver, respectively. The amino acid sequence alignment showed that all 5 clones had critical differences in CDR2-VH and CDR3-VH domains, which might be a key factor affecting their binding capacities to PDL1. The results in this study are beneficial for further development of immunotherapy to disrupt PD-1/PD-L1 interaction.