The depth of mixing layer is one of the important parameters which cannot be assigned a constant value affected by many factors in the slope runoff. The objective of this study was to investigate the effect of slope length and underground biomass on slope runoff, solute transport processes, as well as mixing layer depth. In this study, the experimental plots with the four slope lengths (5, 10, 15, and 20 m) and a width of 2 m were built on the slope with the gradient of 20°. In addition, the plots with the millet or wheat planting were built on the slope. The change of runoff and solute transport was analyzed through simulated rainfall experiments and then to estimate mixing layer depth. The results showed that the runoff rate decreased and more runoff seeped into the slope soil with increasing slope length. Increasing underground biomass also promoted greater rainfall infiltration into the soil. The increase in slope length increased the concentration of solute in runoff, but more underground biomass reduced the nutrients transported with runoff. The effective mixing depth increased with an increase in slope length, but effective mixing depth decreased with increased underground biomass. The modified expression of the equivalent mixing model under different slope lengths and underground biomass could accurately describe the solute transfer process in runoff when compared with complete mixing model and incomplete mixing model based on exponential functions. This research provided a reference for improving the application of mixing layer models in the slope management.