Green economy is a major them of sustainable development. The application of biological waste in engineering is conducive to green development. This study reveals the effect of recycled eggshell waste on the physical and chemical properties as well as nano-microstructure characteristics of asphalt. The hardness, thermal stability and ductility of asphalt were explored by the penetration, softening point and ductility tests. The distribution and relative content of protons in asphalt were revealed by nuclear magnetic resonance hydrogen spectrum (1H-NMR). The microscopic characteristics of the particle morphology and surface structure of the eggshell powder were explored by scanning electron microscopy (SEM). An atomic force microscope (AFM) was used to analyze the evolution laws of asphalt nano-microstructures. The experiment results indicate that (1) the eggshell waste increases the hardness, thermal stability and reduces the ductility of asphalt; (2) the chemical environment in which the protons of the eggshell waste asphalt are located and the H index have no obvious changes; (3) the eggshell powder is characterized by a rough, wrinkled, porous and loosened structure; (4) the nano-microstructure of eggshell waste asphalt exhibits “bee-like structures”, and the different proportion of eggshell waste changes the maturity, size and quantity of the “bee-like structures” and roughness, which can be attributed to the interaction of the asphaltene-waxiness system.