Two-dimensional correlation spectroscopy (2D-COS) can extend the dynamic spectrum to two dimensions, which contains synchronous and asynchronous maps. It not only can separate overlapping peaks to enhance resolution, but also discriminate variation orders of different components. 2D-COS heterospectrum can combine two different spectra into one spectrum, which is applied to trace the relationship between different bands, and identify complementarityof component variations. Synchrotron fluorescence spectroscopy (SFS), Fourier transform infrared (FTIR) and two-dimensional correlation spectroscopy (2D-COS) were applied to analyze the structural composition of dissolved organic matter (DOM) in Wuliangsuhai Lake, trace the covariance between DOM components and functional groups, and reveal their spatial variations. According to the FTIR spectroscopy, DOM was mainly composed of CO, C—H, N—H, C—O functional groups. DOM contained protein-like (PLF), microbial humic-like (MHLF), fulvic-like (FLF) and humic-like (HLF) components by the SFS, among which the MHLF was the dominated component. The decreasing order of the contents of fluorescent materials, PLF and MHLF abundance was north>south>central. The decreasing of the relative FLF abundance was south>north>central, but the relative abundance of HLF kept stable. According to the SFS 2D-COS, the PLF variation was larger than the MHLF in the northern region, whose order was PLF→MHLF; the MHLF showed the negative correlation with the PLF in the central region, whose variation order was PLF→MHLF; the FLF variation was larger than the PLF and MHLF in the southern region, whose order was PLF→FLF→MHLF. According to the FTIR 2D-COS, the C—O was positively correlated to the C—H and CO in the northern region, whose variation order was C—O→C—H→CO; the C—O exhibited the positive correlation with the N—H and CO in the central region, whose variation order was C—O→N—H→C—H→CO; the C—O was the positive relationship with the N—H and CO in the southern region, whose variation order was C—O→C—H→CO. According to the 2D-COS between SFS and FTIR, the C—O showed the positive correlation with the PLF in the northern region; the C—O, N—H and CO had the positive correlations with the MHLF in the central region, but had the negative correlations with the PLF; the C—O, C—H, N—H and CO exhibited thepositive relationship with the FLF. In summary, SFS and FTIR combined with 2D-COS can be used as an effective method to analyze the covariance of DOM components and functional groups in water and reveal their spatial variation.