Raman nanoparticle probes are a potent class of optical labels for the interrogation of pathological and physiological processes in cells, bioassays, and tissues. Herein, we review the recent advancements in fluorescent and Raman imaging using oligodeoxyribonucleotide (ODN)-based nanoparticles and nanostructures, which show promise as effective tools for live-cell analysis. These nanodevices can be used to investigate a vast number of biological processes occurring at various levels, starting from those involving organelles, cells, tissues, and whole living organisms. ODN-based fluorescent and Raman probes have contributed to the achievement of significant advancements in the comprehension of the role played by specific analytes in pathological processes and have inaugurated new possibilities for diagnosing health conditions. The technological implications that have emerged from the studies herein described could open new avenues for innovative diagnostics aimed at identifying socially relevant diseases like cancer through the utilization of intracellular markers and/or guide surgical procedures based on fluorescent or Raman imaging. Particularly complex probe structures have been developed within the past five years, creating a versatile toolbox for live-cell analysis, with each tool possessing its own strengths and limitations for specific studies. Analyzing the literature reports in the field, we predict that the development of ODN-based fluorescent and Raman probes will continue in the near future, disclosing novel ideas on their application in therapeutic and diagnostic strategies.