Abstract: Background and Objective: A 35 kDa of FK506-binding protein of Plasmodium knowlesi (Pk-FKBP35) is a member of peptidyl prolyl cis-trans isomerase (PPIase) consisting of an N-terminal catalytic domain (FKBD) and C-terminal tetratricopeptide repeat domain (TPRD). This study aimed to investigate the thermal stability of full-length Pk-FKBP35 and its domains. Materials and Methods: Full-length Pk-FKBP35 and its isolated domain (Pk-FKBD and Pk-TPRD) were overexpressed in Escherichia coli BL21(DE3) and purified. Thermal stability of purified protein was measured based on the fluorescence signal of 8-anilino-1-naphthalenesulfonic acid (ANS) at the temperature ranging from 25-95°C. Results: The thermal denaturation curve for Pk-FKBP35 shows a cooperative transition with a Tm of 56.49±3.05°C, whereas calculated Tm for Pk-FKBD and Pk-TPRD were 60.67±2.81 and 53.64± 4.21°C, respectively. Higher stability of Pk-FKBD might be due to the high content of β-sheet secondary structure. Thermal unfolding profiles of Pk-FKB35 and Pk-FKBD in the presence of PPIase substrate were considerably different as compared to that of in the absence of the substrate, which might be due to structural stabilization by the substrate. Conclusion: The thermal stability property of Pk-FKBP35 is characterized by two events of interdomain destabilization and substrate-mediated stabilization.