As a technique to improve processing efficiency of defect inspection by quickly adjusting a position of a detection system, provided is a defect inspection apparatus including: a stage that moves with a sample and a pattern substrate placed thereon; an illumination optical system that irradiates an object on the stage from a direction inclined from the normal direction of the pattern substrate; a first detection optical system that detects scattered light in the normal direction; a second detection optical system that detects scattered light in a direction different from the scattered light detected by the first detection optical system; a signal processing unit that processes both scattered light signals; and a control unit. The control unit implements first adjustment processing of adjusting a focal position of the first or the second detection optical system and a three-dimensional position of each detector with respect to an illumination region by using a scattered light signal and second adjustment processing of adjusting a focal point by changing a position in an optical axis direction of a detector of the first or the second detection optical system and a position in a height direction of the stage, and implements the second adjustment processing at a higher frequency than the first adjustment processing.