Straw and cattle manure are common agricultural wastes, and their composting plays a critical role in regional nutrient cycling and organic carbon management. During composting, the structural evolution and humification processes of dissolved organic matter (DOM), fulvic acid (FA), and humic acid (HA) are regulated by environmental factors such as temperature and pH. However, systematic studies on the multi-component fluorescence characteristics of DOM in straw–manure systems and their coupling with environmental variables remain limited. In this study, maize straw and cattle manure were used as raw materials, with four mixing ratios (T1–T4: 2:8, 4:6, 6:4, and 8:2), to investigate the effects of raw material proportions on the structural evolution of DOM, fulvic acid (FA), and humic acid (HA) during composting. Three-dimensional fluorescence spectroscopy combined with parallel factor analysis (EEMs-PARAFAC) was applied to characterize organic components, their transformation patterns, and their relationships with environmental factors. The EEMs-PARAFAC identified 3, 2, and 3 components for DOM, FA, and HA, respectively. Moderate straw–cow manure ratios (T2 and T3) maintained high microbial activity while promoting humic-like component accumulation and FA-to-HA conversion. Fluorescence indices indicated mixed substrate-derived and microbial sources for DOM, predominantly microbial origins for FA, and a shift in HA from substrate-derived to mixed sources. Overall, humification remained low (humification coefficient < 1.5), reflecting an early composting stage. Mantel analysis and partial least squares path modelling (PLS-PM) revealed temperature as the dominant factor associated with HA formation, whereas an alkaline pH inhibited humification. These findings clarify how substrate ratios regulate humification via environmental microhabitats, providing a theoretical basis for optimizing straw–manure co-composting and enhancing compost stability and soil carbon sequestration.