The invention discloses a no-reference structural sharpness-based automatic focusing method. The method includes the following steps that: step S1, grayscale processing is performed on an original image; step S2, image preprocessing is performed with a downsampling method; step S3, low-pass smoothing filtering is performed on a downsampled image, so that a reference image is obtained; step S4, overlapped blocking is performed on the downsampled image and the reference image according to a set block size and a moving step size, so that image sub-blocks are obtained; intra-block variances are calculated, a threshold parameter is set, and image sub-blocks of which the intra-block variances are greater than the threshold are selected; step S5, SSIM values are calculated for the selected imagesub-blocks corresponding to the down-sampled image and the reference image sequentially, and the image sharpness evaluation value, namely NRSS value, of the original image is calculated; and step S6,the NRSS value is converted into a voltage signal, and a lens receives the voltage signal and thereafter completes real-time automatic focusing. The method of the invention has strong robustness, saves calculation time, and well satisfies the real-time requirement of micro-circulation monitoring for automatic focusing.