Early and accurate diagnosis and treatment of cancer depend on rapid, sensitive, and selective detection of tumor cells. Current diagnosis of cancers, especially leukemia, relies on histology and flow cytometry using single dye-labeled antibodies. However, this combination may not lead to high signal output, which can hinder detection, especially when the probes have relatively weak affinities or when the receptor is expressed in a low concentration on the target cell surface. To solve these problems, we have developed a novel method for sensitive and rapid detection of cancer cells using dye-doped silica nanoparticles (NPs) which increases detection sensitivity in flow cytometry analyses between 10- and 100-fold compared to standard methods. Our NPs are ∼60 nm in size and can encapsulate thousands of individual dye molecules within their matrix. We have extensively investigated surface modification strategies in order to make the NPs suitable for selective detection of cancer cells using flow cytometry. The NPs are functionalized with polyethylene glycol (PEG) to prevent nonspecific interactions and with neutravidin to allow universal binding with biotinylated molecules. By virtue of their reliable and selective detection of target cancer cells, these NPs have demonstrated their promising usefulness in conventional flow cytometry. Moreover, they have shown low background signal, high signal enhancement, and efficient functionalization, either with antibody- or aptamer-targeting moieties.