Mueller matrix polarization imaging system (MMPIS) is one of the most prospective tools that can provide a highresolution image of polarization properties for samples or systems. The MMPIS is composed of a laser source, polarization state generator (PSG), the sample, polarization state analyzer (PSA), a high-resolution imaging optics, collimating optics, and a CCD camera. Usually, the traditional eigenvalue calibration method (ECM) can be used to calibrate PSG and PSA. However, the imaging and collimating optics are not calibrated in MMPIS. For the highnumerical-aperture imaging system, the imaging and collimating optics can behave as polarization aberration modifying the tested sample’s polarization properties leading to the erroneous judgment which affects the measurement accuracy of the MMPIS. In this paper, the multi-step eigenvalue calibration method (MECM) is explored to calibrate MMPIS. For the MECM applied to calibrate MMPIS, the calibration samples are required to place in different positions of the light path and the ECM is adopted in each position. In this way, the Mueller matrices of PSG and PSA, as well as the Mueller matrices of imaging optics and collimating optics can be obtained through calculation. To evaluate the measurement accuracy of MMPIS, the sample with known polarization properties such as air is measured. The experimental results show that before calibrating the imaging optics and collimating optics the measurement accuracy of MMPIS is 0.0124, while after the measurement accuracy has been improved to 0.0046, which is 62.90% better than before. The MECM can be used for the requirements of high accuracy measurement.