The liver has complex microenvironments, where parenchymal hepatocytes and non-parenchymal cells coexist. Hepatocytes exhibit different metabolic functions depending on their location by the oxygen gradient and the transcriptional changes of genes, which is called liver zonation. Three-dimensional (3D) liver tissue engineering has reproduced the complex microenvironments, but there is a limitation in analyzing them by location. In this study, a novel 3D tissue-level hepatic cell culture platform is developed via stacking the manipulable collagen sheets to spatially analyze the reconstructed metabolic zonation. The controlled assembly of the sheets containing hepatocytes and endothelial cells, respectively, creates a 3D co-culture environment that improves hepatic function. In addition, the sheet micropatterning can be used to control the accessibility of oxygen and nutrients in the stacked sheets. The disassembly of the stacked sheets enables a layer-by-layer analysis and allows us to confirm the metabolic zonation qualitatively. A demonstration of acetaminophen-induced liver injury using the stacked sheets shows the improved drug sensitivity by co-culture and chemical induction and presents the quantitative results of the different cellular responses to the drug by layers according to metabolic zonation. Therefore, this platform is expected to be used for an in-depth analysis of drug toxicity in complex tissues via spatial analysis.