Soil physicochemical properties and microbes are essential in terrestrial ecosystems through their role in cycling mineral compounds and decomposing organic matter. This study examined the effect of stand age on soil physicochemical properties and microbial community structure in wolfberry ( L.) fields, in order to reveal the mechanism of soil degradation due to long-term stand of . The objective of the study was achieved by phospholipid fatty acid (PLFA) biomarker analysis of soil samples from fields in Zhongning County, Ningxia Province—the origin of . Five stand ages of were selected, < 1, 3, 6, 9, and 12 years (three plots each). The results showed that soil bulk density increased slightly with increasing stand age, while no clear trend was observed in soil pH or total salinity. As the stand age increased, soil organic matter and nutrients first increased before decreasing, with the highest levels being found in year 9. There was an amazing variety of PLFA biomarkers in soil samples at different stand ages. The average concentrations of total, bacterial, fungal, and actinomycete PLFAs in the surface soil initially decreased and then increased, before decreasing with the stand age in summer. The PLFA concentrations of major microbial groups were highest in year 9, with the total PLFA concentrations being 32.97% and 10.67% higher than those in years < 1 and 12, respectively. Higher microbial PLFA concentrations were detected in summer relative to autumn and in the surface relative to the subsurface soil. The highest ratios of Gram-positive to Gram-negative bacterial (G /G ) and fungal to bacterial (F/B) PLFAs were obtained in year 6, on average 76.09% higher than those at the other four stand ages. The soil environment was most stable in year 6, with no differences between other stand ages. Therefore, soil microbial community structure was strongly influenced by the stand age in year 6 only. The effect of stand age on soil G /G and microbial community structure varied with season and depth; there was little effect for F/B in the 20–40 cm soil layer. Principal component analysis revealed no correlations between microbial PLFA concentrations and total salinity in the soil; negative correlations were noted between soil pH and F/B in summer ( < 0.01), as well as between soil pH and fungal PLFA in autumn ( < 0.05). Moreover, microbial PLFA concentrations were correlated with soil organic matter (mean = 0.7725), total nitrogen (mean = 0.8296), total phosphorus (mean = 0.8175), available nitrogen (mean = 0.7458), and available phosphorus (mean = 0.7795) ( 0.01). On the whole, the soil ecosystem was most stable in year 6, while soil organic matter, nutrients, and microbial PLFA concentrations were maximal in year 9; thereafter, soil fertility indices and microbial concentrations decreased and soil quality declined gradually as the stand age increased. Therefore, farmers should reduce the application rate of fertilizers, especially compound or mixed fertilizers, in fields; organic or bacterial manure can be applied increasingly to improve the soil environment and prolong the economic life of .