The intrinsic turbidity of scaffolds formed by natural biomaterials such as collagen fibers prevents high-resolution light microscopy in depth. In this research, we have developed a new method of using light microscopy for penetrative three-dimensional (3-D) visualization of scaffolds formed by collagen, chitosan, or cellulose. First, we applied an optical-clearing solution, FocusClear, to permeate and reduce the turbidity of the scaffolds. The improved photon penetration allowed fluorophores for efficient excitation and emission in the FocusClear solution. Confocal microscopy was applied to achieve cellular-level resolution up to 350 µm for both the fibroblast/collagen and the osteoblast/chitosan constructs and micrometer-level resolution up to 40 μm for the cellulose membrane. The depth of imaging of the cellulose membrane was further improved to 80 μm using two-photon microscopy. Significantly, these voxel-based confocal/two-photon micrographs allowed postrecording image processing via Amira projection algorithms for 3-D visualization and analysis of the scanned region. Although this optical method remains limited in viewing block scaffolds in thin sections, our approach provides a noninvasive way to microscopically examine the scaffold structure, which would be a valuable tool to studying biomaterials and their interactions with the molecule/cell of interest within the scaffold in an integrated fashion.