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The identification of potential diagnostic markers and target molecules among the plethora of tumor oncoproteins for cancer diagnosis requires facile technology that is capable of quantitatively analyzing multiple biomarkers in tumor cells and tissues.Diagnostic and prognostic classifications of human tumors are currently based on western blotting and single-color immunohistochemical methods that are not suitable for multiplexed detection.Herein,we report a general and novel method to prepare single-band upconversion nanoparticles with different colors.The expression levels of three biomarkers in breast cancer cells were determined using single-band upconversion nanoparticles,western blotting and immunohistochemical technologies with excellent correlation.Significantly,the application of antibody-conjugated single-band upconversion nanoparticle molecular profiling technology can achieve the multiplexed simultaneous in situ biodetection of biomarkers in breast cancer cells and tissue specimens and produce more accurate results for the simultaneous quantification of proteins present at low levels compared with classical immunohistochemical technology.