The invention belongs to the technical field of plasma, and discloses a plasma diagnostic method of a multi-amplitude AC bias probe based on a data acquisition card. The plasma diagnostic method comprises the steps of: generating AC bias signals based on the data acquisition card, driving the signals via a power amplifier, and applying the signals to a probe in plasma through a sampling resistor, meanwhile, acquiring a voltage signal on the sampling resistor by means of a differential simulation input port of the data acquisition card commanded by a computer, sending the voltage signal to the computer, and converting the voltage signal into a current signal by the computer; and carrying out spectral analysis and least square method fitting, and further calculating to obtain an accurate electronic temperature value and an accurate ion density value. The plasma diagnostic method solves the problem that electronic temperature and ion density measured by adopting the existing fixed-amplitude AC bias probe diagnostic technique are inaccurate, can diagnose the plasma especially insulating deposition environment plasma, can automatically complete the plasma diagnosis process, and output the accurate electronic temperature and ion density values.