Temperature is an important parameter for many medical and biological applications. It is key to measuring the temperature of acoustofluidics devices for controlling the device’s temperature. In this paper, Rhodamine B was used to measure the temperature change of the microchannel induced by the SSAWs’ thermal effect in microfluidics. A thermocouple was integrated into the microfluidics device to calibrate the relationship between the fluorescent intensity ratios of Rhodamine B and the temperature. Then, the fluid temperature in the microchannel heated by the SSAWs was measured by the fluorescent signal intensity ratio in the acoustofluidics device. The fluid temperature with different input voltages and different flow rates was measured. The results show that SSAWs can heat the still fluid rapidly to 80 °c, and the flow rates will influence the temperature of the fluid. The results will be useful for precisely controlling the temperature of acoustofluidics devices.