Thermotherapy is an attractive alternative to surgery and radiation therapy because of its ability to locally kill tumours while preserving surrounding normal tissues. An important part of successful thermotherapy is real-time temperature monitoring to control the area being heated while protecting normal tissue. The pulsed microwave absorbed by biological tissue can excite ultrasonic waves via thermoelastic expansion, while the magnitude of the acoustic signal is temperature-dependent. The goal of this work is to develop an approach for treatment monitoring of thermotherapy. The pulsed microwave serves as an acoustic excitation source as well as heating source. Temperature is real-time monitored by the magnitude of the thermoacoustic signals. Experiments were conducted in phantoms and fresh ex vivo tissues, an accuracy of 0.2°C was obtained. This approach has the potential to be developed into a viable alternative to current clinical temperature monitoring device for microwave thermotherapy.