An automatic focus detection system for microscopes using the stripe pattern projection method has been developed in this study. Although there are various methods that can be used in the automatic focus detection units of microscopes, the stripe pattern projection method is widely used because automatic focusing can be achieved even on a mirror or rough surface. In the conventional equipment with automatic focusing, the stripe pattern projection unit is set in the illumination unit, and the optical unit for focal position detection is set elsewhere. Therefore, the designer had to develop the automatic focus detection system, when the new equipment was developed. Size and cost reduction for the system are attempted by setting both the stripe pattern projection unit and the optical unit for focal position detection in the commercially available universal illumination unit. An automatic focus detection sensor used for a commercially available camera is adopted and a cylindrical lens is set in front of the sensor. The illuminance of the real image plane is increased and the detection time of the sensor is reduced by one-fifth. As a result of using the objective lens of 0.65 numerical aperture and 40× magnification, the automatic focusing accuracies of ±0.5μm and ±1.0μm have been achieved for samples with a mirror and rough surface, respectively.