Variation of dissolved organic matter (DOM) largely affects the regional and even global carbon cycle. Both optical techniques and ultra‐high‐resolution mass spectrometry have their advantages and disadvantages, and the combination of them can better track DOM cycles. However, the relationship between the optical and molecular characteristics of DOM in the runoff from slopes covered with biocrusts are still unclear. Therefore, optical techniques and Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS) were combined to explore the DOM composition in the runoff from different runoff plots (i.e., bare runoff plot [BP], cyanobacterial crusts covered runoff plot [CP], mixed crusts covered runoff plot [MIP], moss crusts covered runoff plot [MOP]) under different rainfall intensities in the current study. The findings stated that the runoff from BP and CP exhibited a higher proportion of terrestrial DOM under heavy rainfall intensity (HRI), whereas DOM in the runoff from MIP and MOP were primarily controlled by microbial sources. The DOM unique molecules in the runoff from CP were more recalcitrant under HRI than those under light rainfall intensity (LRI), and these differences decreased with the increased with the succession stage of biocrusts. The results of aromaticity characterized by optical method and FT‐ICR MS were generally consistent. Meanwhile, Spearman correlation analysis also indicated that the optical parameters can be utilized for the estimation of molecular characteristics and labile in the runoff from slopes covered with biocrusts after the necessary calibration of the correction between the optical and molecular parameters.