Arid landscapes are shifting from biocrusts- or vascular plant-dominated systems to mosaics where both coexist under climate change; yet, dissolved organic matter (DOM) dynamics in these transitional habitats remain poorly understood. Therefore, three types of runoff plots were set on slopes with biocrusts (BC), Bothriochloa ischaemum (L.) + biocrusts (BIBC), and Artemisia sacrorum Ledeb. + biocrusts (ASBC) in a typical semiarid region. The dynamics of DOM in surface runoff were analyzed by simulated rainfall experiments coupled with optical techniques and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Overall, the DOM loss rates in the BIBC and ASBC were significantly reduced compared to the BC. Runoff from all runoff plots was dominated by the DOM with protein-like components (34% to 96%). Runoff from the BC was characterized by a 1.48- to 2.08-fold higher proportion of humic-like components compared to BIBC and ASBC, accompanied by greater aromaticity and a higher degree of humification. DOM in runoff across all runoff plots was dominated by CHO compounds (52%), with highly unsaturated compounds as the primary constituents (52%). DOM in the runoff from the BC exhibited more stable molecular structures and stronger aromaticity, whereas those from the ASBC showed a higher proportion of multi-heteroatomic components such as CHONP and CHONS. In conclusion, herbaceous vegetation and biocrusts form composite patches that regulate DOM transport and loss by modifying its quantity, composition, and stability. These processes have important implications for water quality and carbon cycling in arid landscapes. This study also highlights the synergistic value of integrating optical and molecular techniques to advance understanding of ecosystem-scale DOM dynamics.