The past few decades have witnessed the rapid development of responsive probes as tools for sensing and imaging of biomarkers in situ in real-time. Among various responsive probes, “dual-key-and-lock” probes have been recently developed as an emerging approach for the detection of biomolecules in specific cell organelles and/or diseased tissues, such as tumours. In comparison with most commonly used “single-key-and-lock” probes, the new “dual-key-and-lock” probes offer substantial advantages in biosensing and imaging, such as high accuracy and precision, minimal “false positive/negative” results, and high selectivity for biomolecule detection at targeted sites. This focus article presents recent advances in the development of “dual-key-and-lock” responsive probes for biosensing and imaging. Two approaches for the design of these probes, including “dual-key-and-one-lock” and “dual-key-and-two-lock”, are highlighted, along with discussions of the challenges and future research directions at the end.