The invention provides a calibration method of a tire uniformity detection device, and belongs to the field of tire uniformity detection. A sensor is fixed on a loading wheel spindle, a loading wheel is in a free state and keeps still, an initial deviation amount of a system is measured firstly, then, a calibration tray, a hook and weights are mounted on the outer edge of the loading wheel, a support rod is used for supporting the tray and the hook, and data are measured for one time while one weight is added every time. Radial sensitivity of the system is obtained by calculating voltage data and given load capacity by a least square method. A side calibration method is consistent with a radial calibration method, the weights are directly added in the side direction, the data are measured for one time when the weight is added every time, and then corresponding side-direction sensitivity is calculated. Herein, a radial and side coupling phenomenon can be verified as well, 100 kg of force is applied on a part above the system in the side direction, the change of radial voltage is measured, and the variable quantity is nearly zero, and therefore, the system does not have the radial and side coupling phenomenon. According to the invention, the calibration structure is simpler, the calibration method is simple and easy, the cost is lower, and the economy is very high. The data measured are fitted by the least square method, and therefore, the accuracy of the obtained result is higher.