This paper presents the design, fabrication, numerical simulation, and experimental validation of a micromachined probe that measures thermal conductivity of biological tissues. The probe consists of a pair of resistive line heating elements and resistance temperature detector sensors, which were fabricated by using planar photolithography on a glass substrate. The numerical analysis revealed that the thermal conductivity and diffusivity can be determined by the temperature response induced by the uniform heat flux in the heating elements. After calibrating the probe using a material (agar gel) of known thermal conductivity, the probe was deployed to calculate the thermal conductivity of Crisco. The measured value is in agreement with that determined by the macro-hot-wire probe method to within 3%. Finally, the micro thermal probe was used to investigate the change of thermal conductivity of pig liver before and after RF ablation treatment. The results show an increase in thermal conductivity of liver after the RF ablation.