Quantum dots (QDs) probes were used to tag and trace cancer biomarkers in cancer tissues based on the system of home-made Hadamard transform (HT) spectral microscopic imaging, which can be applied to provide high-resolution fluorescence spectrum and image of single cells and tissues. In situ fluorescence imaging for cancer marker proteins, such as estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), proliferating cell nuclear antigen (PCNA) and cytokeratin 20 (CK20) in tumor tissues, were realized by using the HT system to capture quantitative information for these proteins when tumor tissues were immunostained with QDs probes. A method to evaluate tumor malignancy of the specimens based on in situ analysis of distribution of marker proteins was proposed based on the comparative study of positive samples and negative controls. The investigation of ER contents of the cores in breast cancer tissue microarrays (TMAs) shows that the technique of QDs-immunohistochemistry (IHC)/HT spectral imaging is more sensitive than conventional IHC method. The results also demonstrate that the QDs-IHC/HT spectral imaging technique can be applied to visualize and quantitatively measure the subcellular molecules inside tumor tissues, and the coupling of HT spectral imaging to the probing of subcellular molecules with QDs has great potential in biology and medical diagnosis.