A polarization calibration method for a multi-spectral aperture-divided simultaneous detection system was introduced. According to characteristics of the detection system, the main factors causing polarization effect were analyzed, including polarizer extinction ratio, polarizer orientation angle deviation induced by assembly error and absolute spectral response. The system detection matrix with polarizer extinction ratio, angle deviation and absolute spectral response was derived. Experiments were designed to solve the unknown calibration data in the system detection matrix. The polarization calibration method and the calibration data were verified with a light source with adjustable polarization degree. The results showed that the measured polarization degrees were 0.74%, 0.01%, 0.80% and 0.59% when the polarization degree of input polarized light was 20% at 490, 670, 870, 1610 nm, which meets the requirement for actual polarization measurement precision in detection system. The proposed polarization calibration method is effective and feasible. The study may contribute to important basis for the practical application of the detection system.