Acquiring information on the precise distribution of a tumor is essential to evaluate intratumoral heterogeneity. Conventional hematoxylin and eosin staining, which has been used by pathologists for more than 100 years, is the gold standard of tumor diagnosis. However, it is difficult to stain entire tumor tissues with hematoxylin and eosin and then acquire the three-dimensional distribution of cells in solid tumors due to difficulties in the staining and rinsing. In this paper, we propose a modified hematoxylin and eosin staining method, in which delipidation and ultrasound waves were applied to enhance tissue permeability and accelerate dye diffusion. This improved hematoxylin and eosin staining method is termed iHE (intact tissue hematoxylin and eosin staining). We applied the iHE method to stain intact organs of mice, which were then sectioned and imaged sequentially. The results showed that the whole tissue was stained homogeneously. Combined with micro-optical sectioning tomography (MOST), the iHE method can be used for 3D volume imaging and to evaluate the intratumoral heterogeneity of the entire tumor tissue spatially. Therefore, this method may help to accurately diagnose the invasion stage of tumors and guide clinical treatments.