Heterogeneity is a common feature of complex biological systems. The heterogeneity across multiple levels collectively drives a variety of (patho)physiological processes. Conventional biosensing approaches for characterizing molecular and cellular heterogeneity, however, are often limited due to the requirement of a large number of cells. Furthermore, existing single cell analysis techniques often require physical isolation or lysis of cells to “snapshot” RNA and protein biomarkers in a small subset of cells. Features of the complex microenvironment, such as hierarchical organization and dynamic cellular processes, are inherently lost by studying cells in isolation, fixation, and lysis. To address this grand challenge in biomedical research, we are developing a nanobiosensing platform based on DNA nanostructures and metallic nanoparticles to enable dynamic single cell gene expression analysis in patient-derived samples. Along with tumor biomanufacturing and 3D imaging strategies, we apply the single cell nanobiosensing platform for probing collective invasion of bladder cancer. Our results reveal the functional implications of tumor heterogeneity in muscle invasive bladder cancer, which will guide the development of prognostic and therapeutic strategies in the future.