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The Amazon Forest is an important and primordial ecosystem, as it controls the dynamics of soil organic matter (SOM) and is considered one of the crucial carbon reservoirs in the world. Based on that, this study evaluated the molecular properties of humic (HA) and fulvic (FA) acid from Amazon soil, with and without Cu(II) ions, by Time-Resolved Laser Fluorescence Spectroscopy (TRLFS) and Fluorescence Spectroscopy (FS). Fluorescence excitation-emission matrix (EEM) was obtained by FS, and EEM spectra were treated by canonical polyadic/parallel factor analysis (EEM-CP/PARAFAC). The results showed that the humification index (A 465nm) determined by FS is directly dependent on fluorophores' CII/CI ratio in these soils with depth (r = 0.89), especially for HA samples in deep horizons, which demonstrated that the CII consists more complex and humified structure of the SOM. The lifetime decay showed the presence of static quenching in the humic fractions. From the lifetime data provided by TRLFS and the different correlations with carbon content and A 465nm , it can be suggested that the fluorescence suppression modifies the molecular properties of the humic fractions with the addition of Cu(II) ions, such as altering the mobility and availability of the metal in the complexation reactions in these Amazonian Spodosols, mainly for the fulvic fraction. • Humification index is directly related on Fluorophores CII/CI ratio in Amazonian Podzols. • Amazonian Podzols showed static quenching. • Copper(II) complexation reaction is dependent on the carbon amount in the Amazonian Podzols. • The lifetime fluorescence showed an excellent spectroscopic indicator for soils's molecular changes. [ABSTRACT FROM AUTHOR]