Dissection of the anatomical information at the single-cell level is crucial for understanding the organization rule and pathological mechanism of biological tissues. Mapping the whole organ in numerous groups with multiple conditions brings the challenges in imaging and analysis. Here, we describe an approach, named array fluorescent micro-optical sectioning tomography (array-fMOST), to identify the three-dimensional information at single-cell resolution from multi-samples. The pipeline contains array embedding, large-scale imaging, post-imaging staining and data analysis, which could image over 24 mouse brains simultaneously and collect the slices for further analysis. With transgenic mice, we acquired the distribution information of neuropeptide somatostatin neurons during natural aging and compared the changes in the microenvironments by multi-component labeling of serial sections with precise co-registration of serial datasets quantitatively. With viral labeling, we also analyzed the input circuits of the medial prefrontal cortex in the whole brain of Alzheimer’s disease and autism model mice. This pipeline is highly scalable to be applied to anatomical alterations screening and identification. It provides new opportunities for combining multi-sample whole-organ imaging and molecular phenotypes identification analysis together. Such integrated high-dimensional information acquisition method may accelerate our understanding of pathogenesis and progression of disease in situ at multiple levels.