In this paper, the influence study of crystal structure on the acoustoelastic stress measurement of bolts was conducted. Three kinds of testing samples with different crystal structures of 45 # medium carbon steel were prepared by different heat treatment processes, and a new design method of piezoelectric ultrasonic transducer which could simultaneously excite both longitudinal and transverse ultrasonic waves was proposed. The acoustoelastic measurement experimental system was established for the prepared samples testing. The ultrasonic transmission time was accurately calculated by cross-correlation method, and the acoustoelastic effects of different crystal structures were analyzed. The experimental results showed that 1) the sensitivity of longitudinal wave is better than that of transverse wave, 2) the crystal structure of the bolt had a great influence on the linearity and repeatability of acoustoelastic measurement, which can be improved by applying the stress cycles to samples, 3) among the three crystal materials prepared in this paper, the medium carbon steel material mainly composed of martensite is the most suitable for acoustoelastic stress measurement.