A flexible temperature microsensor based on a polyimide thin-film substrate is presented. The polyimide thin-film substrate can achieve good contact with skin during tumor ablation, and it has a high ultrasonic propagation rate when ultrasound is passed though the thin substrate. A serpentine structure of platinum film is designed as the sensing element, and the sensor is fabricated using microfabrication technology. The characteristics of the sensor were studied in the temperature range from 25 to 100 °C. The testing results show that there is a good linear relationship between resistance and temperature, and the temperature coefficient of resistance (TCR) of the flexible temperature microsensor is 2035 ppm/°C. The current–voltage (I–V) curve shows that the temperature can be precisely detected when the drive current is lower than 20 mA. Impedance testing results show that impedance does not change with the frequency of the AC current. In addition, the impedance of the sensor under AC conditions has the same value as the resistance under DC conditions, indicating that the performance measured under DC current is the same as that measured under AC current. Such a flexible microsensor can be used to measure the temperature distribution for high-intensity focused ultrasound (HIFU).