Accurate and rapid autofocus technology plays a crucial role in various fields, including automatic optical inspection technology, bio-chips scanning, and semiconductor manufacturing. The current photoelectric autofocus methods have limitations because of detecting the focal plane solely at the center of the microscope field of view. In the application of Stereo-seq the risk of autofocus errors will be increased, which have reduced the robustness of the system, like when the surface of the tested samples are wrinkling and inconsistent thickness, or the detection spot is at the edge of the sample. To enhance the robustness of the autofocus system and mitigate the constraints of the photoelectric autofocus methods, the laser-based arrayed spots photoelectric autofocus method has been proposed. To achieve the uniform light splitting, a 2D-Dammann grating is incorporated into the optical path of the autofocus system, resulting in the formation of an n × n arrayed spots on the surface of the sample. Through experimental verification, it has been demonstrated that this method can achieve the autofocus range of ±100μm and the autofocus accuracy of ±1/4 DOF when applied to a microscope equipped with a 10× objective lens, thereby satisfying the requirements for microscopic focusing. The arrayed light autofocus method devised in this study presents what we believe is a novel research concept for active autofocus detection and holds significant application value.