The spatial composition of the components of a biological system is essential for its proper functioning. The technologies that investigate the spatial context within the tissue has improved enormously in the last decades. Combining with the H&E staining, immuno- histochemistry staining, or fluorescence-based in situ hybridization, novel methods with spatiotemporal omics capabilities have been invented, enabling whole transcriptomics analysis on tissue sections. However, there are still many aspects of these spatiotemporal omics technologies that need to be improved. Here we describe a protocol for a novel technology, termed Spatial enhanced resolution omics-sequencing (Stereo-seq), that enables subcellular resolution and centimeter level field of view. Stereo-seq is based on spatially resolved DNA nanoballs to achieve spatial barcoding. Unlike other spatiotemporal technologies using barcode printing or beads-based substrates, Stereo-seq loads DNA nanoballs onto the photolithography slides and uses MGI Tx Sequencers to decode the location and spatial barcode of each nanoball. This creates the Stereo-seq chip with 500 nm center-to-center pitch size with a 3600 mm2 capture area. After tissue embedding and cryo-sectioning, the 10 μm cryosection slide can be placed onto the Stereo-chip and followed by permeabilization, reverse transcription, cDNA release and sequencing. The spatial gene expression matrix can be generated by mapping the spatial coordinate ID with sequencing data. We demonstrated that the Stereo-seq method can be applied to multiple areas, including transcriptional landscape profiling of mouse embryo organogenesis and macaque brain. In the mouse embryo organogenesis study, we studied the directionality and kinetics of RNA expression variations in a spatiotemporal manner and gained biological insights into the molecular basis of cell type identification with localizations. In the macaque brain study, we utilized Stereo-seq to quantify molecular fingerprints for neocortical layers and subregions, paving the way for better understanding of the mammalian brain structure and function. We believe Stereo-seq represents a powerful spatially resolved transcriptomics solution that enables redefinition of organ composition, disease progression, embryo development and organism evolution