Future climatic change is likely to increase the occurrence of soil freeze–thaw (FT) events in high latitude and/or high altitude zones, which can substantially influence the dynamics of dissolved organic matter (DOM) released into the soils. However, it is not clear how the quantity and quality of soil DOM respond to changing FT patterns under different soil moisture and litter manipulation conditions in northern temperate forest stands. In this study, litter-amended and non-amended forest soils were incubated for 360 days at three soil moisture levels (30%, 60%, and 90% water-filled pore space) and three intensities of FT disturbance (low, high, and none). We quantified heterotrophic respiration, enzyme activity, microbial biomass, and water-extractable organic matter (WEOM) as a proxy for DOM in soils during the incubation experiment. The quality of WEOM was characterized by biodegradability, UV absorbance and parallel factor analysis modelling of fluorescence excitation emission matrices. Concentrations of water-extractable organic carbon (WEOC) and biodegradable WEOC declined continuously in all treatments over the 360-day incubation period. The dominant component of fluorescent WEOM shifted from humic- and fulvic- like components during the first 108 days of incubation, to protein-like components of microbial origin, characterized by high aromaticity, at the end of the incubation period. Litter amendment, FT disturbance, and their interaction increased WEOC concentrations in soils during the early 108-day incubation period, particularly in soils with low moisture and high FT intensity, but these increases disappeared after 252 days incubation at 15 °C. Litter-derived fluorescent WEOM in soils without FT disturbance was dominated by protein-like components after 14 days of incubation, but these were replaced by humic- and fulvic-like components after 108 days. This replacement effect was weaker in soils with FT disturbance, which we attribute to changes in microbial properties, including enzyme activity, microbial biomass and activity.