The step-temperature technique can be used to measure bio-tissue thermal physical parameters. During the derivation of the step-temperature technique, the measuring probe was assumed to be made of single thermistor sensing element, and the electrical power was assumed to be uniformly distributed throughout the probe bead. However, the probe bead in reality is made of multi-layer structure, and the electrical power is not uniformly distributed throughout the probe bead. In this paper, a mathematical model is built for the step-temperature technique for multi-layer structure probe bead, and a numerical simulation is made to analyze the effects of probe configuration. The physical meaning of the calibrated values of the probe parameters (bead radius and bead thermal conductivity) is also explained theoretically. The results show that the step-temperature technique is still valid for the measurement of bio-tissue thermal physical parameters for the multi-layer structure thermistor probe bead on condition that the bead radius and the bead thermal conductivity are obtained through pre- calibration.