Self-propelled nanomotors offer considerable promise for developing novel biosensing protocols involving ‘on-the-fly’ recognition events. This article reviews recent advances in using catalytic nanomotors for bioaffinity sensing and for isolating target biomolecules and cells from complex biological samples. A variety of receptors, attached to self-propelled nanoscale motors, can thus move around the sample and, along with the generated microbubbles, lead to greatly enhanced fluid transport and accelerated recognition process. Such operation addresses the challenges imposed by the slow analyte transport in designing sensitive bioaffinity assays. The recognition element can be attached onto the motor surface or embedded in the motor material itself. Receptor-functionalized nanomotors based on different biomolecular interactions have thus been shown extremely useful for rapid target isolation from complex biological samples without preparatory and washing steps. Tubular microengine microtransporters, functionalized with antibody, ss-DNA, aptamer or lectin receptors, are particularly useful for direct detection and isolation of proteins, nucleic acids, proteins or cancer cells. Micromotors with ‘built-in’ recognition, exploiting the selective binding properties of the outer layer of such micronegines, can also be used. Greatly enhanced analyte–receptor interactions can also be achieved through the increased fluid transport associated with the movement of unmodified micromotors. The attractive features of the new motion-based bioaffinity sensing and separation protocols open up new opportunities for diverse biomedical, environmental and security applications. Bioaffinity sensing based on receptor-functionalized microengines. Display omitted •Bioaffinity sensing based on receptor-functionalized microengines.•Motion-based biosensing.•'On-the-Fly' recognition events.