In this paper, we propose a simple yet effective method for estimating defocus blur of microscopy images. The defocus blur at each edge point is estimated directly from local gradient profiles. To reliably detect edges and estimate blur in noisy and blur images, the image gradient is computed at multiple scales. The defocus blur at an edge point is firstly computed by the gradient ratio between different scales and is then recomputed by estimating the variance of the Gaussian gradient profiles. The proposed method is robust to noise and edge mis-localization. Experimental results on synthetic and real microscopy images demonstrate the effectiveness of our method.