Due to the tool-workpiece separation effect, high-speed ultrasonic vibration cutting has the proven ability to reduce tool flank wear and prolong tool life by up to approximately three times when turning Ti-6Al-4V alloys in the recent research. Temperature reduction caused by the cooling medium penetrating into the gaps of the tool-workpiece contact zone is considered to be the primary reason for this remarkable performance. In this paper, a tool-workpiece thermocouple system was developed for the temperature measurement of high-speed ultrasonic vibration cutting Ti-6Al-4V alloys. First, the calibration of the tool-workpiece thermocouple was conducted to identify the piecewise linear relation between thermoelectric power and cutting temperature. Next, the insulation of the thermoelectric circuit from both the vibration-stimulated circuit and the experiment platform was realized. Finally, comparative cutting experiments on high-speed ultrasonic vibration cutting and conventional cutting with and without cutting fluid were carried out. The experimental results demonstrate that in the dry cutting experiments, no obvious temperature difference between these two methods occurred. However, when cutting fluid was applied, compared to conventional cutting, a maximum 30% temperature reduction could be realized by high-speed ultrasonic vibration cutting with a duty cycle value of 0.55 at cutting speeds of 250 to 300 m/min. Therefore, cutting fluid is thought to be an essential part of effective cooling in the process of high-speed ultrasonic vibration cutting and its cooling behavior during such a short separation interval needs further researches.