In order to accurately obtain the refocusing image of the depth position according to the parameter, a calibration method based on the step ruler is proposed. Taking the original image by the light field camera, the image sequence is obtained by the refocusing technique, and the scale surface of the image sequence is calculated by the definition evaluation function, to find the clearest image, complete calibration of the refocusing parameter. Evaluating the calibration results, the knifeedge method is used to calculate the Gaussian defocusing parameter of the refocused image. The value of this method is reduced by more than 25% compared with the method of the scale ruler, which can effectively improve the accuracy of refocusing of the light field camera. The sectioning imaging experiment was carried out, and the sectioning refocusing images under the two calibration methods were compared to verify that the refocusing results of the step ruler calibration are clearer and suitable for the light field optical sectioning tomography system.