As a biocompatible and bioactive natural tissue engineering scaffold, porcine acellular dermal matrix (PADM) has limitations for the application in tissue regeneration due to its low strength and rapid biodegradation. Here, purified PADM was modified by a nontoxic cross-linker (genipin) to enhance its mechanical properties and improve its resistance to enzymatic degradation. In vitro testing results demonstrated that the stiffness of the genipin cross-linked PADM was improved and biodegradation rate was decreased. Results of cell proliferation assays showed that the cross-linking reaction by genipin did not undermine the cytocompatibility of PADM. Furthermore, genipin cross-linking imparted an observable fluorescence allowing visualization of the scaffold's three-dimensional (3D) porous structure and cell distribution by confocal laser scanning microscopy (CLSM). Immunostaining of the cell nuclei and cytoskeleton indicated that MC3T3-E1 preosteoblasts were tightly adhered to and uniformly distributed onto the cross-linked PADM scaffold. Results of this study suggest that the 3D porous genipin cross-linked PADM with intrinsic fluorescence may have broader applications for tissue engineering scaffolds where higher mechanical stiffness is needed.