Abstract: The composition, content, and turnover rate of soil dissolved organic carbon (DOC) directly affect the composition and activity of microbial communities, thus influencing the mineralization process of soil organic carbon. This study investigated the effects of conservation tillage practices on the fluorescence properties of soil DOC in different regions to provide a theoretical basis for the scientific selection of tillage methods. In this study, four conservation tillage treatments, including no-tillage (NT), no-tillage + straw returning (NR), subsoiling (ST), and subsoiling + straw returning (SR), were set up in Keshan, Heilongjiang Province (thick-layer black soil) and Gongzhuling, Jilin Province (thin-layer black soil). Traditional tillage (CT) and tillage + straw returning (CR) were used as controls. Fluorescence spectroscopy was used to analyze the content, composition, and structural characteristics of farmland soil DOC in 0–20 cm of thick-layer black soil and thin-layer black soil under different tillage treatments. The results showed that in the thick-layer black soil area, the soil TOC content was SR>NR>CR>ST>NT>CT, and the DOC content was SR>ST>NT>NR>CR>CT. Both soil TOC and DOC contents were the highest in the SR treatment and were significantly increased (P<0.05) by 6.88% and 25.61%, respectively, compared to those in the CR treatment. In the thin-layer black soil area, the TOC and DOC contents in the NR treatment were the highest, and the DOC content significantly increased (P<0.05) by 21.30% compared to that in CR. Parallel factor analysis showed that DOC in the thick-layer black soil and thin-layer black soil areas was divided into three components: C1 was composed of tryptophan proteins, C2 was a fulvic acid-like substance, and C3 was a humus-like substance. Fluorescence index analysis indicated that thick-layer and thin-layer black soil areas were affected by both internal and external influences. In the thick-layer black soil area, the SR treatment had the lowest autogenic characteristics, which were greatly affected by exogenous input, with the highest humification coefficient and a more stable DOC structure. In the thin-layer black soil area, the NR treatment had the lowest autochthonous source characteristics and the lowest microbial contribution, mainly from exogenous straw input, with a stable DOC structure not easily utilized by microbial decomposition. In conclusion, conservation tillage measures enhanced both TOC and DOC in both thick- and thin-layer black soil regions of agricultural fields. The SR treatment was more conducive to the accumulation of TOC and DOC and the stabilization of carbon pools in the thick-layer black soil, and the NR treatment was more advantageous for the enhancement and stabilization of organic carbon in the thin-layer black soil.