Construction of self-illuminating semiconducting nanocrystals, also called quantum dots (QDs), has attracted much attention recently due to their potential as highly sensitive optical probes for biological imaging applications. Here we prepared a self-illuminating QD system by doping positron-emitting radionuclide Cu-64 into CdSe/ZnS core/shell QDs via a cation-exchange reaction. The Cu-64-doped CdSe/ZnS QDs exhibit efficient Cerenkov resonance energy transfer (CRET). The signal of Cu-64 can accurately reflect the biodistribution of the QDs during circulation with no dissociation of Cu-64 from the nanoparticles. We also explored this system for in vivo tumor imaging. This nanoprobe showed high tumor-targeting ability in a U87MG glioblastoma xenograft model (12.796 ID/g at 17 h time point) and feasibility for in vivo luminescence imaging of tumor in the absence of excitation light. The availability of these self-illuminating integrated QDs provides an accurate and convenient tool for in vivo tumor imaging and detection.