A new method of microlens focal length measurement by rotation method based on grating diffraction is introduced. When parallel rays travel through the grating, various angle-order diffracted rays will appear behind the grating because of grating diffraction. According to the grating equation, the angle between the ±1 st-order and the 0-order diffracted rays is determined by grating period and testing wavelength. Taking a camera nearby the focus of microlens to get the picture one by one, from clarity function of the picture, the focus position will be decided. In addition, the distance of the +1 st-order and the 0-order spot center can be tested from the picture. With the angle and spot central distance between the ±1 st-order and the 0-order, the focal length can be measured. The measurement result shows that this method has a good precision for focal length measurement of microlens. And because of its high efficiency, this method can also be used for focal length of microlens array measurement.