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The calibration of camera systems employing zoom lenses raises several challenges, as changes in lens control settings introduce various imaging conditions, especially different degrees of defocus. This article presents a Fourier-based defocus calibration method to calibrate a camera attached with an electronically focus-tunable lens (ETL). Different from the traditional method which requires changing the scale of the calibration object for sharp images, the proposed method is more concerned with the frequency characteristics of the captured images than the gray distributions. According to the invariance of the fundamental frequencies before and after defocusing the images, coordinates of feature points can be extracted accurately, even in different degrees of defocus. Therefore, one normal size target can also be qualified for zoom system calibration. Then polynomial approximation is used to account for the variation of the intrinsic and distortion parameters across the zoom range. Extensive simulations and experiments were implemented to demonstrate the proposed idea’s robustness against defocus, flexibility, and accuracy.