Accurate profiling of microscopy images from small scale to high throughput is an essential procedure in basic and applied biological research. Here, we present Microsnoop, a novel deep learning-based representation tool trained on large-scale microscopy images using masked self-supervised learning. Microsnoop can process various complex and heterogeneous images, and we classified images into three categories: single-cell, full-field, and batch-experiment images. Our benchmark study on 10 high-quality evaluation datasets, containing over 2,230,000 images, demonstrated Microsnoop's robust and state-of-the-art microscopy image representation ability, surpassing existing generalist and even several custom algorithms. Microsnoop can be integrated with other pipelines to perform tasks such as superresolution histopathology image and multimodal analysis. Furthermore, Microsnoop can be adapted to various hardware and can be easily deployed on local or cloud computing platforms. We will regularly retrain and reevaluate the model using community-contributed data to consistently improve Microsnoop.