Abstract: 3D dose distribution measurement in radiotherapy based on scintillator light field imaging was achieved by 3D reconstruction of scintillation light produced by the scintillator energy deposition. The refocusing position of the image obtained by the light field camera affected the accuracy of the scintillation light 3D reconstruction. A method of refocusing position calibration using checkerboard calibration plate was presented. The checkerboard calibration plate in a known position was captured by the Lytro Illum light field camera, and the focus stack sequence was obtained by the light field data processing. The Gaussian defocusing parameter σ of all refocusing images was obtained by the knife-edge method, and the refocusing parameter α corresponding to the minimum σ is obtained. Finally, the corresponding relationship between some α and refocusing position was given to complete the refocusing position calibration. Compared with the commonly used scale calibration method, the Gaussian defocusing parameter can be reduced by 2 % to 10 %, which is helpful to improve the measuring accuracy of the 3D dose in radiotherapy.