A great deal of melanin was found in the waste alkali liquor produced by extraction of chitin from Catharsius molossus L. Discarding the lye could harm the environment and cause waste of resources. In this paper, melanin from C. molossus L. was recovered through add precipitation and purified by pepsin and so on. The purity, chemical composition and structure of the prepared melanin were explored by UV-visible absorption spectroscopy, Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, high resolution C-13 Cross polarization magic angle spinning nuclear magnetic resonance spectroscopy pyrolysis gas chromatography mass spectrometry, X ray diffraction, X ray fluorescence, matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry, thermal analysis, and so on. The results showed that the purity of the prepared melanin was higher than the commercial standard melanin and it was a kind of nanoaggregates composed of a large quantity of 5,6-dihydroxyindole eumelanin and a small amount of phaeomelanin. In addition, the prepared melanin was irregular in shape and its structure could be divided into three levels: advanced structure maintained by polypeptides, substructure maintained by the ferric ion and microstructure. In particular, the smallest structural unit showed the graphite-like layered structure containing five layers linked by non-covalent bonds and each layer mainly consisted of 5,6-dihydroxyindole and its derivatives, which might be connected to each other through various chemical bonds. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.