Abstract: Radiofrequency ablation is a widely used minimally invasive technique for tumor treatment in clinical practice, where precise control of thermal field distribution is essential to ensure both therapeutic efficacy and safety. The thermal properties of biological tissues are the key determinants of the temperature distribution within a patient’s body during tumor ablation therapy. Accurate measurement of these properties is crucial for advancing research and clinical applications of radiofrequency ablation technology. The current national standards specify multiple methods for measuring thermal properties of various materials, yet lack standardized methodologies specifically for biological tissues. This paper outlines the fundamental principles of biothermal parameter measurement and investigates a dual-probe thermal pulse technique based on the cylindrical-perfect-conductor model. Through systematic analysis of measurement errors from both modeling and experimental perspectives, the study addresses a critical research gap in error characterization for this methodology. The results of this paper demonstrate that probe spacing and probe length are the main factors affecting measurement errors in the dual-probe thermal pulse method for measuring thermal properties of biological tissues. Taking the measurement of liver tissue thermal parameters as an example, probes with lengths of 30mm and 20mm introduce volumetric heat capacity errors of 4.78% and 7.08% respectively, while a 2% variation in probe spacing results in 3.93% error in volumetric heat capacity measurement and 3.67% error in thermal diffusivity measurement. This study indicates that in practical measurements of biological tissue thermal properties, the measurement and control of probe spacing significantly impact result accuracy, and it is recommended to use reference materials to calibrate probe spacing to improve final measurement precision.