The extracellular environment is an architectural support for tissue cells and stem cells, which is very important in cell adhesion, migration and differentiation. In this study, we prepared a self-assembled macromolecular matrix, naming it the preosteoblast-derived matrix (PDM). The primary focus was to characterize PDM in component and structure, and then to evaluate its osteogenic potential as a two-dimensional (2D) microenvironment. Preosteoblasts were cultured on a coverslip and then decellularized using a cocktail solution of detergents and enzymes, leaving a matrix without the cellular components. The surface of the PDM had a fibrillar mesh structure, as imaged by scanning electron microscope (SEM). The compositions of PDM, fibronectin, type I collagen, and laminin were identified using immunofluorescent staining. Adjustment of culture time or cell seeding density produced not only different compositional disparity in quantity, but also showed distinct pattern of macromolecule assembly. F-Actin staining revealed that early cell morphology was quite different as the type of substrates changed. Preosteoblasts were much more elongated on PDM to a certain direction and soft in their adhesion. Cells were proliferating faster in PDM as compared to the coverslip (control) or the gelatin-coated surface. When they were cultured for 2 weeks in three different substrates, von Kossa staining exhibited that calcium deposits were much densely formed over PDM. This result was also quantitatively supported by calcium assay. Measurement of alkaline phosphatase (ALP) activity demonstrated the positive effect of PDM, with higher ALP activity than the other groups. The present study indicates that naturally derived macromolecular matrix is able to carry major protein components as well as a fibrillar structure and that it may provide preosteoblasts with a favorable surface microenvironment for osteogenic differentiation.