Cellular function depends on the spatial organization of cells and biomolecules within the tissue microenvironment. Advances in spatial omics have enabled profiling of molecular features such as transcriptome, proteome and epigenome, and there has been rapid progress of imaging-based approaches to study spatial three-dimensional (3D) genome organization. Here we present Spatial-ATAC-Hi-C, a microfluidic‑based platform for genome-wide, spatially resolved joint-profiling of 3D genome organization and chromatin accessibility on tissue slides. Applied to mouse and human brains, Spatial-ATAC-Hi-C revealed distinct chromatin architecture and gene regulatory programs in neuronal and non-neuronal populations in their native tissue context. In glioblastoma and astrocytoma samples, we detected spatially resolved 3D genome alterations, copy number variations and structural variations across tumor regions, revealing clinically relevant oncogenic events and clonal heterogeneity. By co-profiling of genome architecture and chromatin accessibility while preserving tissue architecture, Spatial-ATAC-Hi-C provides a powerful tool for studying spatial gene regulation in human biology and disease.