Abstract: Rice and crayfish co-cultivation is the largest cultivation mode in China and is an important part of ecological agriculture. However, in the process of cultivation, the activities changed the physical and chemical properties of field soils, which had a significant effect on the change in organic matter. Dissolved organic matter (DOM), one of the most active components of organic matter, is also the subject of modification, but there are few related studies on the characteristics of modified dissolved organic matter. Therefore, in this study, ultraviolet-visible (UV-Vis) absorption spectra and three-dimensional fluorescence spectra were used to calculate different spectral parameters combined with parallel factor analysis (PARAFAC) to study the characteristics of soil DOM under four rice and cultivation modes (integrated rice-crayfish rotation system with ring groove, RS₁; integrated rice-crayfish system with ring groove, RS₂; integrated rice-crayfish system, RS₀; traditional rice monoculture, MR). The results revealed that there were weak shoulder peaks at 260–280 nm in the UV-Vis absorption spectrum of soil DOM in all modes, and the absorbance decreased with increasing wavelength and gradually approached zero. The UV-Vis absorption spectral parameters SUVA₂₅₄ (specific absorption coefficient) and UV₂₅₄ (UV absorbance at wavelength 254 nm) of soil DOM in different rice and crayfish cultivation modes (RS₁, RS₂ and RS₀) were lower than those of traditional rice monoculture (MR). The E2/E3 (ratio of UV absorbance at 254 nm to 365 nm) value was smallest in RS₀, and the S_R (spectral slope ratio) values of the two co-cropping patterns (RS₂ and RS₀) were averaged from 0.5 to 2.0. For all modes, the mean value of fluorescence index >1.9, humification index < 4, biological index = 0.6−0.7. All rice and crayfish cultivation modes had resolved two protein-like components (C1, C3) and two humic-like components (C2, C4), C1 accounted for a higher proportion and was negatively correlated with other components, and the remaining components were positively correlated with each other. The principal component analysis identified four effective components, which were arranged according to the contribution rate: PC1 represented the concentration and aromaticity of DOM; PC2 represented the biological characteristics, molecular weight and degree of humification of DOM; PC3 represented the protein components and autogenic features in DOM; PC4 represented the molecular structure of DOM. The results showed that many unsaturated aromatic structure molecules existed in the soil DOM of all modes. Rice and crayfish cultivation reduced the degree of humification and aromaticity of soil DOM. The RS₀ mode had the largest molecular weight of soil DOM and certain exogenous characteristics; the soil fertilizer retention ability was better, and the comprehensive benefit was the best among the three rice and crayfish farming modes. There was no significant difference in the material composition of soil DOM under different modes, which was related to the indirect effects of agricultural management practices. The source of C1 was different from that of other components, which may be related to the decomposition and transformation of DOM by microorganisms. Additionally, the difference in flooding was one of the main reasons for the difference in soil DOM characteristics; the soil releases DOM and DOC through flooding, which significantly affects the characteristics of soil DOM. In production practice, attention should be paid to the application of organic fertilizers, which can maintain soil fertility, increase DOM and microbial diversity, maintain soil fertility, and monitor the status of contaminants in soil and crops on farmlands.