Molecular subtyping of cancer can greatly help to understand the development of disease and predict tumor behavior. Exploring detection methods for precise subtyping is appealing to prognosis and personalized therapy. During the past decades, DNA‐based biosensors have exhibited great potential in cancer diagnosis due to their structural programmability and functional diversity. Despite the encouraging progress that has been made, there remains an issue in improving the accuracy and sensitivity of cancer subtyping due to the complex process of disease, especially in preclinical or clinical applications. To accelerate the development of DNA sensors in the identification of cancer subtypes, in this review, we summarized their advances in molecular subtyping by analyzing the heterogeneity in categories and levels of biomarkers between cancer subtypes. The strategies toward genomic and proteomic heterogeneity in cells or on the cell surface, as well as the cancer excretions including extracellular vesicles (EVs) and microRNA (miRNAs) in serum, are summarized. Current challenges and the opportunities of DNA‐based sensors in this field are also discussed. Molecular subtyping of cancer is crucial for understanding the development of disease and predicting tumor behavior. This review focuses on the advanced strategies of molecular subtyping using DNA‐based biosensors for the identification of cancer subtypes based on different biomarkers, including cells, extracellular vesicles (EVs), and microRNAs (miRNAs) in serum.