Multi-functional Hadamard transform spectral microscopic imaging system was employed to provide high-resolutional fluorescence spectrum and image of tiny samples such as single cells and tissues. Semiconductor quan-tum dots (QDs) have several photo-physical advantages such as broad excitation spectrum, multi-color fluorescence with one single wavelength light source, narrow fluorescence emission peak, high photostability and long fluores-cence lifetime, which make QDs good markers of the fluorescence spectral imaging microscopic analysis in biomedical applications. Based on immunostaining with quantum dots (QDs) emitting at 610 nm to tag and trace two breast cancer biomarkers human epidermal growth factor receptor2 (HER2) and estrogen receptor(ER) in human breast cancer tissue with in situ fluorescence imaging, sensitive spectra and images were obtained. Moreover, by compa-ring the differences of fluorescence spectra and 4D images between positive samples, (the human breast cancertis-sues) and negative control, (the normal human breast tissues) by Hadamard transform spectral microscopic imaging system, a method to evaluate tumor malignancy of breast cancer tissues based on the analysis of distribution of HER2 and ER was proposed. The results show that the Hadamard system can be applied to visualize and quantitatively measure the subcellular proteins such as HER2 and ER inside the tumor tissues. The method developed with the above technique can be applied to quantitatively evaluate tumor malignancy and is advantageous over conventional method.