Aimed at the problem of automatic focus and image system quality evaluation in microscopy imaging, a micro-image definition evaluation method is presented by combining multi-scale decomposition tools and absolute gradient operators. The multiscale and multidirectional non-subsampled Shearlet transform is utilized to decompose the input micro image into a low frequency sub-band image and a number of high frequency sub-band images. Combined with the anti-noise threshold setting, the gradient absolute sum values of each sub-band image were calculated. By using the different effects of image sharpness on the low-frequency and high-frequency sub-band coefficients, the ratio of the high-frequency to low-frequency gradient absolute value operator was taken as the final evaluation value of the microscopic image sharpness. The simulation experiment and actual experiments were car-ried out and the experimental results illustrated that the proposed approach has good monotonicity and anti-noise characteristics. Compared with other classic evaluation algorithms, the presented method obtained superior per-formance on sensitivity, stability and robustness. It has very good practical application values.