Abstract: The optical performance of the microscope objective lens directly affects the quality of microscopic imaging, so it is of great significance to detect its wavefront aberration. A method for detecting the aberration of a microscopic objective lens based on a Hartmann wavefront sensor was proposed. This method employed the two-sphere method to calibrate systematic errors, enabling aberration characterization of microscope objectives. The model was established and simulated and analysed, and the results show that measured wavefront aberrations closely matched actual values, verifying model validity. The experimental setup was constructed, and the aberration detection of the microscope objective was carried out. The measurement shows the objective lens's root mean square (RMS) detection accuracy is ≤ 10 nm, with a repeatability accuracy of < 0.3 nm. This confirms the practical feasibility of the detection model. The method is simple and easy to set up, providing an detection model for wavefront aberration detection of medium and low power finite-conjugate microscope objectives.