Abstract: In the process of microwave thermotherapy for small biological tissue, the microwave energy of thermotherapy antenna is focused too much, the treatment range is beyond the target area, and other healthy tissues are burned. The antenna structure is composed of a round copper sleeve shell and two circular microstrip patches welded on both sides of the flange of SMA connector. The dielectric substrate of microstrip patch is FR-4 material with relative permittivity of 4. 4, and the thickness of substrate is 1. 6 mm. The measured reflection coefficient of the antenna at 915 MHz is -18. 5 dB. In the experiment, a small pork tissue of 40 mm×40 mm×40 mm is heated by the antenna, and the heating condition of the antenna is tested by two temperature measurement methods, i. e. infrared thermal imager and Thermocouple thermometer. The results show that the effective treatment area of the antenna is a semi-ellipsoid of 20 mm×20 mm×10 mm, and the temperature distribution is relatively uniform. It is suitable for the application of small volume in vitro microwave thermotherapy system.