DNA-functionalized quantum dots (DNA-QDs) have found considerable application in biosensing and bioimaging. Different from the first generation (I-G) DNA-QDs prepared via conventional bioconjugation chemistry, the second generation (II-G) DNA-QDs prepared via one-step DNA-templated QD synthesis features a defined number of DNA valencies (usually monovalency), which is preferable for controlled assembly and biological targeting. In this review, we summarize recent progress in designing QD probes based on II-G DNA-QDs for advanced sensing and imaging applications. It opens up new avenues for highly sensitive and intelligent sensing of a range of disease-relevant biomolecules in vitro and in living cells.