OTF of under test lens was tested with pinhole image processing and analyzing, the influences of sampling frequency on test precision were analyzed, and a calibration method of frequency conversion scale and sampling frequency on image plane was presented. The test results on 50 mm focus standard lens showed that the accurately calibration and import of system setting parameter effect on test precision directly, calibration method in this paper are accurate and reliable, and can acquire parameter of effecting on spatial frequency calibration and OTF of lens under test accurately, without knowing subsystem parameter.