At present, extracting water-soluble organic matter (WSOM) from agricultural organic waste is primarily used to evaluate soil organic matter content in farmland. However, only a few studies have focused on its vertical behavior in the soil profile. This study aims to clarify the three-dimensional fluorescence spectrum characteristics of the WSOM samples in 0–60 cm black soil profile before and after different chemical fertilizer treatments after six years of fertilization. Fluorescence spectroscopy combined with fluorescence and ultraviolet-visible (UV-Vis) spectroscopies are used to divide four different fertilization types: no fertilization (T0), nitrogen phosphorus potassium (NPK) (T1), biochar (T2), biochar + NPK (T3), and biochar + N (T4) in a typical black soil area. The vertical characteristics of WSOC are also analyzed. The results showed that after six years of nitrogen application, T2 had a significant effect on the fluorescence intensity of Zone II (decreasing by 9.6% in the 0–20 cm soil layer) and Zone V (increasing by 8.5% in the 0–20 cm soil layer). The fluorescent components identified in each treatment group include ultraviolet radiation A humic acid-like substances (C1), ultraviolet radiation C humic acid-like substances (C2), and tryptophan-like substance (C3). As compared with the land with T1, the content of C2 in the 20–60 cm soil layer with T2 was lower, while that of C2 in the surface and subsoil with T3 was higher. In addiiton, there were no significant differences in the contents of C1, C2, and C3 by comparing the soils applied with T3 and T4, respectively. The composition of soil WSOM was found to be significantly influenced by the addition of a mixture of biochar and chemical fertilizers. The addition of biochar alone exerted a positive effect on the humification process in the surface soil (0–10 cm). NPK treatment could stimulate biological activity by increasing biological index values in deeper soil layers (40–50 cm). Nitrogen is the sovereign factor that improves the synergism effect of chemical fertilizer and biochar during the humification process. According to the UV-Vis spectrum and optical index, soil WSOM originates from land and microorganisms. This study reveals the dynamics of WSOC in the 0–60 cm soil layer and the biogeochemical effect of BC fertilizer treatment on the agricultural soil ecosystem.