Interpreting the mechanisms and principles that govern gene activity and how these genes work according to -their cellular distribution in organisms has profound implications for cancer research. The latest technological advancements, such as imaging-based approaches and next-generation single-cell sequencing technologies, have established a platform for spatial transcriptomics to systematically quantify the expression of all or most genes in the entire tumor microenvironment and explore an array of disease milieus, particularly in tumors. Spatial profiling technologies permit the study of transcriptional activity at the spatial or single-cell level. This multidimensional classification of the transcriptomic and proteomic signatures of tumors, especially the associated immune and stromal cells, facilitates evaluation of tumor heterogeneity, details of the evolutionary trajectory of each tumor, and multifaceted interactions between each tumor cell and its microenvironment. Therefore, spatial profiling technologies may provide abundant and high-resolution information required for the description of clinical-related features in immuno-oncology. From this perspective, the present review will highlight the importance of spatial transcriptomic and spatial proteomics analysis along with the joint use of other sequencing technologies and their implications in cancers and immune-oncology. In the near future, advances in spatial profiling technologies will undoubtedly expand our understanding of tumor biology and highlight possible precision therapeutic targets for cancer patients. Keywords: immuno-oncology; proteome; spatial profiling technologies; transcriptome; tumor heterogeneity.