Ecological restoration in the Loess-hilly region is a useful method to improve soil quality and reduce erosion. Soil microbes and aggregates are of great important for soil quality improvement during the processof eco-restoration. However, the eco-recovery effect on microbial biomass and diversity in soil aggregates is unknown. The objectives of thisproject were to use phospholipid fatty acid (PLFA) analysis (1) to studysoil microbial biomass and diversity among aggregate size fractions under three land uses (natural succession grassland [N.G.], Caragana korshinskii shrubland [C.K.], and cropland [Cr.]) in China’s Loess Plateau and (2) to evaluate variation of microbial community structure within different aggregate sizes (>5, 3–5, 2–3, 1–2, 0.25–1, and <0.25 mm) using principal component analysis. Our results showed that the N.G. and C.K. restoration can increase soil microbial biomass relative to Cr. soil, which was supported by the physiological index changes of microbes in different aggregate size classes. Soil aggregate of 0.25 to 1 and >5 mm comprised approximately 60% to 70% of the total aggregates. In the grassland soil, the concentration of organic carbon, total nitrogen, and total PLFAs were significantly highest (P < 0.05) in the 1- to 2-mm aggregate class. In similarly sized aggregate samples from different land uses, the bacterial, Gram-positive bacteria, actinomycete PLFAs and the total PLFAs in the Cr. soil were significantly lower (P < 0.05) than in N.G. and C.K. soils. Based on principal component analysis, we observed that the 1- to 2-mm aggregate fraction is most active in terms of microbial biomass and contains the largest amount of nutrients in cropland, whereas in shrubland this was the case for the 3- to 5-mm fraction. Therefore, we conclude that different aggregate fractions are essential for improving soil quality depending on land use type.