During the past 50 years, the amount of agricultural fertilizer used in Northern China increased from about 7.5 kg ha−1 in the 1950s to approximately 348 kg ha−1 in the 1990s. Given that little is known about the effects of nitrogen fertilization on soil labile carbon fraction in Northern China, this paper evaluated such effects in terms of microbial biomass and dissolved organic carbon in the Sanjiang Plain located in Northeast China. Soils with different cultivation time and undisturbed marsh with Deyeuxia angustifolia were selected to study the effects of nitrogen fertilization on the soil labile organic fractions microbial C (biomass C, microbial quotient, and basal respiration) and to estimate the contributions of nitrogen input on the dynamics of soil labile carbon. Continuous nitrogen application decreased total organic and dissolved organic carbon concentrations significantly, leading to the lack of carbon source for microbes. Therefore, continuous nitrogen fertilizer application induced negative effects on measured soil microbiological properties. However, a moderate nitrogen application rate (60 kg N ha−1) stimulated soil microbial activity in the short term (about 2 months), whereas a high nitrogen application rate (150 kg N ha−1) inhibited measured soil microbiological properties in the same period.