Changes in environmental conditions could alter the composition structure and chemical properties of dissolved organic matter (DOM), then affect its biogeochemical cycling processes. In this study, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectrum, and Fourier transform infrared spectroscopy (FTIR) combined with two-dimensional correlation spectroscopy were applied to evaluate the effect of pH on DOM characteristics and its interaction with Cu2+. (1) Our results suggested that the fluorescence intensity of different DOM components increased remarkably along with the pH value increasing from 5 to 10. Humic-like components showed the most significant changes, and fulvic-like components responded fastest to pH changes. It was caused by pH changes induced exposure of some functional groups, such as carbonyl, phenolic, and carboxyl. (2) Two-dimensional correlation analysis of fluorescence spectra revealed that changes in pH could significantly affect the Cu2+-binding capacity of different DOM components but could not affect the binding sequence to Cu2+ with DOM. Three fluorescent components were identified by parallel factor analysis of the three-dimensional fluorescence spectrum. The nonlinear fitting of quenching curve for fluorescent components quantitatively verified our results. (3) FTIR results showed that, under hinger pH conditions, DOM has more binding sites and stronger binding affinities with Cu2+. The structural change of DOM upon Cu2+ addition under pH 5 and 10 conditions followed the order ofpolysaccharide C—O>phenols>aldehyde and ketone CO>aromatic C—H and polysaccharide C—O>amide II C—N>phenols>aliphatic C—H>aldehyde and ketone CO>carboxyl C—OH>aromatic C—H>carboxyl CO, respectively. Furthermore, two-dimensional hyperspectral correlation analysis of fluorescence spectra and FTIR indicated that humic-like fractions of DOM participated in the Cu2+binding after the phenolic and aryl groups.