Multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) are developed for simultaneous targeting and imaging of cancer cells in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer, and linking this polymer to tumor-targeting ligands, such as antibodies and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mouse show that the QD probes can be delivered to tumor sites by both enhanced permeation and retention (passive targeting) and by antibody binding to cancer-specific cell surface biomarkers such as prostate-specific membrane antigen (active targeting). Using both subcutaneous injection of QD-tagged cancer cells and the systemic injection of multifunctional QD probes, multicolor fluorescence imaging of as few as 10–100 cancer cells can be achieved under in vivo conditions. The use of spectrally resolved imaging can efficiently remove autofluorescence background and precisely delineate weak spectral signatures in vivo. These results suggest that QD probes and spectral imaging can be combined for multiplexed imaging and detection of genes, proteins, and small-molecule drugs in single living cells, and that this imaging modality can be adopted for real-time visualization of cancer cell metastasis in live animals.