System calibration is fundamental to the overall accuracy of the ultrasonic temperature measurement, and it is basically involved in accurately measuring the path length and the system latency of the ultrasonic system. This paper proposes a method of high accuracy system calibration. By estimating the time delay between the transmitted signal and the received signal at several different temperatures, the calibration equations are constructed, and the calibrated results are determined with the use of the least squares algorithm. The formulas are deduced for calculating the calibration uncertainties, and the possible influential factors are analyzed. The experimental results in distilled water show that the calibrated path length and system latency can achieve uncertainties of 0.058 mm and 0.038 μs, respectively, and the temperature accuracy is significantly improved by using the calibrated results. The temperature error remains within ±0.04°C consistently, and the percentage error is less than 0.15%.