Abstract: Liquid manure is a type of fertilizer derived from the harmless treatment of livestock and poultry waste, applied in liquid form; however, the impact of its continuous application on the content and composition of organic matter in topsoil remains unclear. In this study, soil samples were collected from plots treated with dairy liquid manure (LF) for 1 to 12 years, chemical fertilizer (CF), and no fertilizer (CK). Using three-dimensional fluorescence spectroscopy combined with parallel factor analysis (EEM-PARAFAC), the effects of long-term liquid manure application on soil organic matter and dissolved organic matter were systematically investigated. The results showed that, compared to the CK and CF treatments, the application of liquid manure did not significantly increase the organic matter content in the soil. However, as the duration of liquid manure application increased, a cumulative trend in soil organic matter was observed. The fluorescence spectroscopy results revealed that, compared to the CK treatment, liquid manure application increased the relative content of structurally complex fulvic-like and humic-like components in the soil dissolved organic matter while significantly reducing the relative content of protein-like components. This led to a gradual increase in the humification index from CK to CF to LF treatments, with the index showing a slow increase with extended duration of LF application. The study demonstrates that long-term application of liquid manure can alter the composition of soil dissolved organic matter through direct exogenous input and microbial transformation, increasing the proportion of large-molecular-weight, recalcitrant components in the soil dissolved organic matter. These findings provide valuable insights for guiding the application of liquid manure in agricultural practices in China.