The composition of dissolved organic matter (DOM) is complex, which is an important part of the river biogeochemical cycle and has a good indication of the aquatic systems. The stepped urban landscape river was taken as the research area to study the interaction between DOM composition changes of rivers and aquatic parameters based on synchronous fluorescence spectrum (SFS). The fluorescence region integration, redundancy analysis (RDA), two-dimensional correlation spectroscopy (2D-COS), and principal component analysis (PCA) were used to analyze SFS and aquatic parameters. The SFS was divided into four regions according to the fluorescence characteristics of DOM, i.e. tyrosine-like (TYRLF), tryptophan-like (TRPLF), microbial humic-like (MHLF), and terrestrial humic-like (HLF). The factor of regional integral correction was proposed to improve the accuracy of the semi-quantitative analysis of SFS. The results showed that the DOM in the aquatic systems of the stepped landscape river in summer was mainly composed of biological sources (TYRLF, TRPLF, and MHLF) (> 80%). SFS-2D-COS indicated that the trend of protein-like components (TYRLF and TRPLF) was opposite to that of humic-like components (MHLF and HLF). The order of DOM components change in water was TYRLF > TRPLF > HLF > MHLF. SFS-PCA divides the DOM of the water body according to structural characteristics and realizes the rapid classification and qualitative analysis of DOM samples based on SFS.