The density of collagen fibers are automatically evaluated by the proposed algorithm based on enhanced Frangi filter. In optical virtual biopsy, Second Harmonic Generation (SHG) microscopy has been developed and applied to observe collagen fibers in the dermal layer of the human skin. The density of collagen fibers is a feature to describe the condition of collagen fibers which can provide indicator for the early pathological diagnosis and aging characteristic in SHG images. The proposed algorithm is capable of objectively and effectively reflecting that the distributions of collagen fibers are close or loose in virtual biopsy images as revealed by the experimental results. Moreover, the proposed method provides insight into identifying the correlation between the early symptoms of diseases and the density of collagen fibers.