In order to study the effects of the long-term application of compost on the source and composition characteristics of soil DOM in fluvo-aquic soil, this study took the experimental field of long-term application of compost at Quzhou Experimental Station in Hebei Province as the research object. It used three-dimensional fluorescence spectroscopy to explore the differences of the source and composition characteristics of DOM in the soil profile under long-term application of the high dosage of bio-compost (EMI), the conventional dosage of bio-compost (EMII), high dosage of traditional compost (TCI), the conventional dosage of traditional compost (TCII) and chemical fertilizers (CF). The results showed that the distribution of soil dissolved organic carbon (DOC) in soil profiles under different fertilization treatments was quite different, and the application of compost significantly increased the DOC in the 0~20 and 60~80 cm soil layers by 81.94%~171.33% and 61.18%~152.18%, respectively. The fluorescence spectrum index showed that the source of DOM is a mixed source of microorganisms and plants. The increase of compost dosage will increase the degree of DOM’s humification, causing the surface soil’s DOM to migrate from land source to biological source. As the soil depth increased, DOM migrated from land to biological sources. Three-dimensional fluorescence spectroscopy and fluorescence area integration showed that bio-compost and traditional compost increased the content of humic acid-like substances, which increased with the increase of application rate. High-dosage bio-compost and traditional compost increased fulvic acid-like and soluble microbial byproduct-like substances. The application of chemical fertilizers and compost reduced the content of tryptophan-like. The contents of humic acid-like, fulvic acid-like, and soluble microbial byproduct-like substances showed an overall decreasing trend with soil depth; Tyrosine-like protein increased with soil depth; Tryptophan-like substance first increased and then decreased with the increase of soil depth, and the content of tryptophan-like substance was the highest in 20~40 cm. Correlation analysis showed that soil physical and chemical indexes such as TP, TN, CEC, AK, SOC and DOC were negatively correlated with tyrosine-like substances, positively correlated with fulvic acid-like substances, soluble microbial products and humic acid-like substances. Moreover, NO-3-N, TN, pH and SOC were significantly positively correlated with tryptophan-like substances. In a word, long-term application of compost increased the DOM content in the surface layer of fluvo-aquic soil and significantly changed the composition of DOM in the soil and the distribution characteristics on the profile.