To assist unattended fundus examination equipment in automatically performing fundus examinations under different lighting environments, an automatic pupil center location and alignment device was developed and a circle approximation algorithm was proposed to automatically locate the pupil center. First, an image was preprocessed by binarization and contour extraction, and a filling algorithm was used to eliminate the influence of noise and speckle for follow-up processing. The circle approximation algorithm proposed in this study was subsequently utilized to accurately locate the pupil center. Finally, the identified location of the pupil center was used to control the stepper motor to move the two-dimensional platform and align the image acquisition and pupil centers. To evaluate the efficiency of the proposed algorithm, the location accuracy and calculation rate were compared with those of the traditional and improved Hough circle algorithms. The precision and average consumption time of the proposed algorithm are 93.33% and 95.67 ms, respectively. Compared to that of the traditional and improved Hough circle algorithms, the precision of the proposed method improve 3.5 and 2 times, respectively, and the average consumption time is reduced by 68.86% and 63.11%, respectively. Experimental results indicate that the proposed system significantly improved the accuracy and calculation rate of automatic pupil location and alignment under conditions in which the illumination parameters cannot be strictly controlled. In summary, the proposed system is able to meet the real-time, accuracy, and robustness requirements of unattended fundus examination equipment, which has great significance for its popularization.