Superconducting quantum interference devices ( SQUID) are superconducting devices that use two properties to resolve very weak magnetic field variations, including flux quantization and Josephson effect. They are used in a wide range of applications for sensitive detection of magnetic signals. Therefore, to ensure the accuracy and quality of its output mapping, it is essential to calibrate it periodically during use. In this article, the calibration system optimization of SQUID second-order magnetic gradiometer in use is discussed. A Cartesian coordinate system is established with the center of the calibration coil as the center point, and the relative position in the Y-axis direction is fixed. The calibration coil is moved from the Z-axis direction to find the maximum position range of current sensitivity. Then, the position is further calibrated precisely to find the relative position in the X-axis direction that is least sensitive to the movement in the Z-axis direction. , which provides a larger tolerance range for possible human errors in calibration. The analytical model and the finite element simulation model are validated against each other to provide a theoretical basis and precedence for subsequent experiments. The calibration coefficient of SQUID second-order magnetic gradiometer in use is determined to be 1. 107 through the analytical model, the finite element model and the measured data. The uncertainty generated by the proposed calibration method is analyzed to provide greater robustness for the calibration of the gradiometer under low-noise environment conditions.