The development of nano-composite materials puts higher demand on morphological analysis techniques. The bio-nano-composite material systems is among the most challenging nano-composite materials for morphology characterization due to its sensitivity to damage, complex molecular conformations and nano-structures. The aim of this project is to provide a nanometer resolution and convenient chemical mapping tool based on scanning electron microscope (SEM) and electron spectroscopy for complex bio-composite systems. This would combine the backscattered electron and secondary electron techniques based on the angle-selection and energy-filtering methods. Theoretical electron behavior in the SEM is calculated using Monte Carlo simulations for reference. This SEM technique is validated and applied on representative artificial and natural bio-composite systems. Poly(N-isopropylacrylamide) (PNIPAM) composite material is a family of widely applied temperature-responsive bio-materials. The phase separation and morphology in PNIPAM nano-composites can affect the bio-compatibility of material system. Silk fiber is a well known natural bio-material with exceptional properties as well as a model hierarchical material system. The organization of nano-repeating unit in silk is expected to be a key factor in the mechanical property formation. Direct chemical mapping of this organization was not available up to now in convenient methods. Our SEM techniques (secondary electron hyperspectral imaging, SEHI) were validated and applied for mapping of these bio-materials and provided high-resolution chemical characterization of their nano-structures. The application of SEM techniques were further extended to different silk fibers and artificial silk materials. Such experiment validated the complex fine structure of secondary electron spectra measured on silk materials. The comparison of the electron spectra in different silk materials suggested a possible reflection of protein conformation in secondary electron spectra and this may be exploited for characterization of such complex materials in future applications. In summary, SEM analysis technique using electron selective detection methods capable of nano-resolution chemical characterization were validated and applied on nano-bio-composite materials. These techniques show great potential for morphological analysis in complex and sensitive composite materials in the future.