To facilitate the selection of effective therapeutic pathways and improve clinical outcomes, sensitive and simultaneous diagnosis of multiple trace biomarkers or cancer cells from complex living samples is particularly critical in the early stages of tumor development. To achieve this, we have combined the selectivity and affinity of aptamers with the spectroscopic advantages of fluorescence resonance energy transfer (FRET) nanoparticles (NPs). This has produced an aptamer-conjugated FRET NP assay that performs simultaneous multiplexed monitoring of cancer cells with the desired degree of sensitivity and selectivity. First, by changing the doping ratio of three different dyes, the FRET-mediated emission signatures could be tuned such that the nanoparticles would exhibit multiple colors upon excitation with a single wavelength. These FRET nanoparticles were then modified by a few aptamers specific for different cancer cell lines, in this case, T-cell leukemia and B-cell lymphoma. As, a result, simultaneous and sensitive detection of multiple cancer cell targets was achieved. Therefore, our aptamer-conjugated FRET NPs are highly promising for potential applications in the sensitive monitoring of multiple cancer cells for biomedical research and medical diagnostics. ; NIH ; NCI ; NIGMS ; State of Florida Center for Nano-Biosensors ; Scientific Research Foundation ; Department d'Universitats, Recerca i Societat de la Informacio' de la Generalitat de Catalunya, Spain