In order to study the spectral characteristics of soil dissolved organic matter (DOM) under agricultural non-point source pollution in the Huaihe River Basin, this study collected the soil from the Pihe River, a first-order tributary of the Zhengyangguan River, and used the parallel factor analysis method (PARAFAC) to obtain two components of humus and protein. Soil dissolved organic carbon (DOC) content was significantly different under different land use types (P<0.01). The soil DOM in the Huaihe River Basin is affected by both terrestrial and microbial sources. The biological index (BIX) of forest soil is significantly larger than that of cultivated land at different depths, which is caused by the rich plant diversity of forest land. The humification degree of cultivated land soil is higher than that of forest land, and the humification effect of cultivated land soil fertility is stronger due to human factors. The humification index (HIX), BIX and fluorescence index (FI) have a good indication of the degree of soil humification. At the same time, the greater the BIX, FI and spectral slope ratio (S_R), the greater the contribution of microorganisms to soil DOM.