The mechanisms of carcinogenesis, cancer invasion and metastasis remain unclear because of the complexity of cancer cells and tumor microenvironment. Thus, the urgent development of a novel approach for cancer detection and real-time monitoring is crucial in order to decipher the intricate molecular information responsible for tumor biological behaviors. Quantum dots (QDs) are engineered fluorescent nanoparticles with unique optical and chemical properties, which have shown a great potential as promising platforms for biomedical applications. Here, we focus on the clinical applications of quantum dot-based nanotechnology in cancer detection and diagnosis, covering topics on individual cancer diagnosis and treatment by in-vitro and in-vivo molecular imaging technologies, sentinel lymph node (SLN) mapping, cancer associated proteins detection in blood, circulation tumor cells (CTCs) detection, and in-depth understanding of the biological behaviors from the perspective of tumor microenvironment. In addition, the major challenges in translating quantum dot-based detection methodologies into clinical applications and promising future directions are also discussed.