To overcome the disadvantages of the existing microscopy autofocus technology, this study proposes a method for detecting the defocusing distance and orientation of a sample according to the distance and direction from the center of the field of view (FOV) of the focusing band in a tilt plane image, which is acquired using a tilt camera. The feasibility of this method is verified by constructing an experimental microscope autofocus system. Results show that this system can quickly obtain a defocusing distance and orientation by detecting the amount of horizontal shift from the center of FOV of the focusing band in a single-frame tilt plane image, which can be determined according to a calibration curve between the defocusing distance and focusing band's horizontal-shift value. Autofocus can then be achieved in applications using motor drive. The optical structure presented herein is simple and cost-effective. It can be directly used in a conventional, trinocular microscope-imaging system and can thus be applied to the autofocus process required in online, in situ, large FOV, and high-resolution detection in various fields.