Abstract • Humification evolution was identified with non-destructive characterization method. • Humification process from precursors to fulvic and humic acid was confirmed. • MnO2 alone had limited oxidation ability to form HA. • MnO2 played a key role as a catalyst to transform FA to HA in the presence of O2. • MnO2 could affect the structure of the humification products. Abiotic humification is important in the formation and evolution of organic matter in soil and compost maturing processes. However, the roles of metal oxides in abiotic humification reactions under micro-aerobic remain ambiguous. The aim of this study was to use non-destructive measurement methods to investigate the role of MnO2 in the evolution of humic substances (HSs) during oxidative polymerization of polyphenol-amino acid. Our results suggested a synergistic effect between MnO2 and O2 in promoting the polymerization reaction and identified that MnO2 alone had a limited ability in accelerating the transformation of fulvic acid (FA) to humic acid (HA), whereas O2 was the key factor in the process. Two-dimensional correlation spectroscopy (2D-COS) showed that the evolution in the UV-vis spectra followed the order of 475–525 nm>300–400 nm>240–280 nm in the humification process, indicating the formation of simple organic matter followed by FA and then HA. 13C nuclear magnetic resonance (13C NMR) analysis revealed that the products under both air and N2 conditions in the presence of MnO2 had greater amounts of aromatic-C than in the absence of MnO2, demonstrating that MnO2 affected the structure of the humification products. The results of this study provided new insights into the theory of abiotic humification.