The objectives of this study were to explore the effects of long-term and continued application of fertilizers and manures on microbial biomass, soil biological activity and their seasonal variations in surface and subsurface soils in relation to soil fertility. For this, soils were sampled in spring, summer and autumn from Shenyang Long-term Experimental Station, northeastern China. The results showed that soil total nitrogen (N), organic carbon (C), basal respiration, microbial biomass and enzymatic activity increased in manure-amended surface soils, but decreased with soil depth. Long-term application of inorganic fertilizers significantly decreased soil pH value, sucrase activity and microbial biomass C, but increased soil metabolic quotient (qCO2). However, no significant effect of inorganic fertilizers on soil total N, urease activity and microbial biomass N was observed in comparison with CK0 (neither tillage nor fertilization) and CK (no fertilizers). There was no significant difference between CK0 and CK in soil total N, organic C and microbial activity in surface soil layer (0–20 cm), but these parameters in subsurface soil layer (20–40 cm) were higher in CK than in CK0. Moreover, seasonal changes were observed in terms of soil nutrient contents, enzymatic activity, microbial biomass and soil respiration. There were significant correlations between soil microbial biomass C and N, between organic C and sucrase activity and between total N and urease activity, respectively. It is recommended that combined use of organic manure with inorganic fertilizers should be considered to maintain higher microbial biomass, soil biological activity and soil fertility. Considering considerably high nutrients reserve and microbial activity in subsurface layers of soil and wind-erosion-caused nutrient loss in spring in north China, we also propose that low tillage should be considered to make use of nutrients in soils.