We report on the development of a nanowire substrate-enabled laser scanning imaging cytometry for rare cell analysis in order to achieve quantitative, automated, and functional evaluation of circulating tumor cells. Immuno-functionalized nanowire arrays have been demonstrated as a superior material to capture rare cells from heterogeneous cell populations. The laser scanning cytometry method enables large-area, automated quantitation of captured cells and rapid evaluation of functional cellular parameters (e.g., size, shape, and signaling protein) at the single-cell level. This integrated platform was first tested for capture and quantitation of human lung carcinoma cells from a mixture of tumor cells and leukocytes. We further applied it to the analysis of rare tumor cells spiked in fresh human whole blood (several cells per mL) that emulate metastatic cancer patient blood and demonstrated the potential of this technology for analyzing circulating tumor cells in the clinical settings. Using a high-content image analysis algorithm, cellular morphometric parameters and fluorescence intensities can be rapidly quantitated in an automated, unbiased, and standardized manner. Together, this approach enables informative characterization of captured cells in situ and potentially allows for subclassification of circulating tumor cells, a key step toward the identification of true metastasis-initiating cells. Thus, this nanoenabled platform holds great potential for studying the biology of rare tumor cells and for differential diagnosis of cancer progression and metastasis.