Humus is the main component of soil organic matter, which is important for soil fertility and nutrient cycling. Forest litterfall as a carrier of nutrients and carbon plays an essential role in the formation of soil humus. The input of litterfall can provide raw materials for the synthesis of soil humus. However, the evidence shows that the incorporation of fresh organic matter into soil can increase microbial activity due to the higher availability of energy released, then stimulate the degradation of soil humus. These conflicted results can lead to a confusing relationship between litterfall and soil humus. Therefore, we carried out a field incubation experiment in three typical subalpine forests (coniferous, mixed and broadleaved forests) on the eastern Qinghai-Tibetan Plateau. There are two treatments in this experiment. One allowed litterfall to enter the soil normally and the other prevented litterfall to enter the soil. Soils were sampled in October (the end of the growing season), March (the end of the winter season), and September (the middle of the growing season) from 2017 to 2019. Litterfall production, and the values of ΔlogK (the ratio of absorbance at 400 to that at 600 nm on a logarithmic scale), E4/E6 (the ratio of absorption at 465 to that at 665 nm), and A600/C ratios of soil extracted humic substances were analyzed in each period. The results showed that, during the two-year experiment, the overall performance of the soil humification degree in the three forests showed the order as coniferous forest>mixed forest>broadleaved forest, and exhibited an obvious dynamic pattern with decreasing in winter and increasing in the growing season. Statistical analysis indicated that litterfall had insignificant effects on the degree of soil humification in the three forests, but the input of litterfall significantly changed the seasonal tendency of soil humification, which was more prominent in broadleaf and mixed forests in winter. The input of litterfall significantly reduced the degree of soil humification in broadleaved and mixed forest in winter, while had no significant effect on the three forest soils in the growing season. These results suggested that the increase of temperature in winter under the condition of climate warming might lead to an increase in the degree of soil humification, and the presence of litterfall could slow the increase proceeding, which are scientifically significant for soil fertility management and sustainable management in subalpine forests due to seasonal frequent freeze-thaw cycles and the sensitivity to climate change.