This paper analyzes the main factors affecting the signal integrity of highspeed data acquisition circuit based on the characteristics and requirements of highfrequency signal test instrument use environment.Aiming at the reflection and crosstalk of highfrequency signal, the signal integrity design is carried out from the multilayer PCB board laminated design, differential signal transmission line serpentine routing and adding impedance matching network, respectively. The network topology model of differential signal transmission line is established, andthe structure and parameters of the impedance matching network are determined through simulation, which reduces the signal reflection and crosstalk caused by transmission line impedance mismatch. The power integrity is analyzed, the circuit ground bounce is reduced with the inner electric layer segmentation and filter capacitor network, and thereby, the system noise of the test instrument is reduced. Finally, test experimentunder simulation environment was conducted, which verifies the signal integrity analysis of highspeed data acquisition circuit; the experiment result shows that with signal integrity design, the antijamming capability of the testinstrument is improved, and the collected data are reliable and effective.