Abstract Understanding complex biological systems requires tracing cellular dynamic changes across conditions, time, and space. However, integrating multi-sample data in a unified way to explore cellular heterogeneity remains challenging. Here, we present Stereopy, a flexible framework for modeling and dissecting comparative and spatiotemporal patterns in multi-sample spatial transcriptomics with interactive data visualization. To optimize this framework, we devise a universal container, a scope controller, and an integrative transformer tailored for multi-sample multimodal data storage, management, and processing. Stereopy showcases three representative applications: investigating specific cell communities and genes responsible for pathological changes, detecting spatiotemporal gene patterns by considering spatial and temporal features, and inferring three-dimensional niche-based cell-gene interaction network that bridges intercellular communications and intracellular regulations. Stereopy serves as both a comprehensive bioinformatics toolbox and an extensible framework that empowers researchers with enhanced data interpretation abilities and new perspectives for mining multi-sample spatial transcriptomics data.