Although freshwater lakes are considered to be an important source of greenhouse gas (GHG) emissions, the potential driving mechanisms of such emissions are not well understood, especially in steppe lakes. In this study, the GHG emission characteristics in Hulun Lake Basin, including Hulun Lake, Beier Lake, Wulannuoer Lake, and their surrounding watersheds were investigated. The average methane (CH4) and nitrous oxide (N2O) emission fluxes released from rivers were 67.84 ± 20.53 and 0.11 ± 0.04 μg m−2·min−1, which were larger than those of lakes, with values of 28.60 ± 13.02 and 0.06 ± 0.02 μg m−2·min−1, respectively. Conversely, the average carbon dioxide (CO2) emission flux from lakes (1816.58 ± 498.98 μg m−2·min−1) was higher than that of rivers of (1795.41 ± 670.49 μg m−2·min−1). The water in Hulun Lake Basin was rich in organic matter and had a high chemical oxygen demand (COD). Three-dimensional fluorescence combined with a parallel factor analysis (3D-EEM-PARAFAC) demonstrated that the organic matter was composed of four humus types (from Component 1 (C1) to Component 4 (C4)), of which, C1 and C4 were terrestrial humus. The fluorescence index (FI) and humification index (HIX) indicated that the organic matter in the water was mainly imported from exogenous humus. The GHG emission fluxes were negatively correlated with these four components, indicating that GHG emissions were mainly affected by the organic matter source and components, and humus was the most important factor that inhibited GHG emissions in steppe lakes. However, the GHG emission flux was relatively high in some areas of the lake, especially in areas with high nutrient levels or where algal blooms occurred, as evidenced by the significantly positive correlations with total nitrogen (TN), total phosphorous (TP), and chlorophyll-a (chl-a) (p < 0.01). The algae-derived organic matter simulated the decomposition of refractory humus, thus, promoting GHG emissions. These findings are crucial for accurately evaluating the GHG emission fluxes, understanding the carbon cycle, and proposing future management strategies for steppe lakes.