Afforestation has been shown to strongly affect substrate stoichiometry and profoundly influence the microbial community. However, the degree to which microbial and activity are linked to soil carbon (C), nitrogen (N) and phosphorus (P) stoichiometry in afforested ecosystems remains unclear. In this study, soil samples were collected from Robinia pseudoacacia L. (RP42, RP27, and RP17) and farmland (FL) sites with a chronosequence of 42, 27, and 17 years. We determined the microbial biomass C (Cmic), N (Nmic), Cmic to organic C (Csoil) ratio (Cmic:Csoil), Nmic to total nitrogen (Nsoil) ratio (Nmic:Nsoil), and metabolic quotient (Rmic:Cmic) to investigate microbial. We also measured soil b-glucosidase (BG), N-acetylglucosaminidase (NAG), acid phosphatase (AP), as well as Csoil, Nsoil, and total phosphorus (Psoil). The results showed that, compared with FL, Csoil:Psoil and Nsoil:Psoil were increased during aggradation, whereas Csoil:Nsoil increased in RP27 and RP17 but decreased in RP42. Cmic:Csoil and Nmic:Nsoil were 94% and 182% higher in RP42 than FL, respectively. However, Rmic:Cmic was lower in RP42, RP27, and RP17 than FL by 64%, 36% and 25%, respectively. Moreover, we found that BG, NAG, AP, BG:NAG, and BG:AP in RP42, RP27, and RP17 were higher than in FL. Compared with global soil (0.62 and 0.13), the lower BG:AP (0.42) and BG:NAG (0.03) ratios in present study indicated that Psoil may be limited. Additionally, redundancy analysis (RDA) revealed that the Csoil: Nsoil was positively correlated with Rmic: Cmic but negatively with Cmic: Csoil and Nmic: Nsoil, while Csoil:Psoil and Nsoil:Psoil were also significantly and negatively correlated with BG:AP, BG:NAG, and NAG. Therefore, our results indicated that afforested ecosystem are highly susceptible to changes in soil microbial and enzyme stoichiometry during aggradation and may become P-limited. Such changes were modulated by soil nutrient stoichiometry.