Integumentary organs exhibit diverse morphologies and functions. The complex mechanical property of the architecture is mainly contributed by the ingenious multiscale assembly of keratins. A cross-scale characterization on keratin integration in an integument system will help us understand the principles on how keratin-based bio-architecture are built and function in nature. In this study, we used feather as a model integument organ. We develop autofluorescence (AF) microscopy to study the characteristics of its keratin assemblies over a wide range of length scales. The AF intensity of each feather component, following the hierarchy from the rachis to barb to barbule, decreased with the physical dimension. By combining the analysis of AF signal and tensile testing, we can probe regional material density and the associated mechanical strength in a composite feather. We further demonstrated that the AF micro-images could resolve subtle variations in the defective keratin assembly in feathers from frizzled chicken variants with a mutation in α-keratin 75. The distinction between AF patterns and the morphological features of feather components across different length scales indicated a synergetic interplay between material integration and complex morphogenesis during feather development. The work shows AF microscopy can serve as an easy and non-invasive approach to study multiscale keratin organizations and the associated bio-mechanical properties in diverse integumentary organs. This approach will facilitate our learning of many bio-inspired designs in diverse animal integumentary organs/appendages.