Detection of circulating tumor cells (CTCs) plays an important role in cancer diagnosis and prognosis. In this study, aptamer-conjugated upconversion nanoparticles (UCNPs) are used for the first time as nanoprobes to recognize tumor cells, which are then enriched by attaching with magnetic nanoparticles (MNPs) and placing in the presence of a magnetic field. Owing to the autofluorescencefree nature of upconversion luminescence imaging, as well as the use of magnetic separation to further reduce background signals, our technique allows for highly sensitive detection and collection of small numbers of tumor cells spiked into healthy blood samples, and shows promise for CTC detection in medical diagnostics.