The anaerobic ammonium oxidation (anammox) process is globally an important nitrogen-cycling process mediated by specialized microbes. However, still little information is documented about anammox microbial community structure under agricultural soils. The anaerobic incubation experiment was conducted to study the impacts of different land use soils fertilized by C-13-urea on the activity and diversity of anammox bacteria using stable isotope to probe the phospholipid fatty acid (PLFA-SIP). The C-13 was preferentially incorporated in ratios PLFAs 16:1 omega 7c, 16:1 omega 5c, and 16:0. The results revealed that the abundance of the anammox bacteria (both hzs-beta and hzo) were observed in vegetable soil V1 and paddy soils (R1 and R2) means that they were positively correlated with C-13-urea but were negatively correlated with NO3 (-)-N and NH4 (+)-N concentrations. Thus, C-13-PLFAs 16:1 omega 7c, 16:1 omega 5c, and 16:0 could be the biomarker as soil anammox. The anaerobic microbial community composition of soils under different land use systems was diverse, and V1, R1, and R2 had similar microbial diversity and higher microbial biomass. The principal component analysis between soil properties and gene abundance suggested that not only pH but also soil organic matter, available P, and available K were important factors for the anammox process. This study suggested that C-13-Urea-PLFA for anaerobic incubation was a simple method to study anammox microbial community structure through affecting the soil nutrients, and the different land use systems played important roles in determining the microbial composition of soils.