Soil extracellular enzyme activities and associated enzymatic stoichiometry are considered sensitive indicators of nutrient availability and microbial substrate limitation. However, many of previous studies have been focusing on uppermost soil layer with a single enzyme as representative of the whole nutrient acquisition, leading to critical uncertainties in understanding soil nutrient availability and its relationship with microbial activities in deeper soils. In the current study, we investigated C-, N- and P-acquiring enzyme activities across a range of soil layers (0-10, 10-20, 20-40 and 40-60 cm), and examined the microbial C, N and P limitation in natural secondary forests (NSF) and Chinese fir (Cunninghamia lanceolata) plantation forests (CPF) in subtropical China. The results showed that microbial C and P co-limitation was detected in the two typical subtropical forests at all soil depths, rather than microbial N limitation. Microbial C and P limitation fluctuated along soil depth, but higher N was demanded by microbes in soil under 20 cm in both forests. The present results highlight the asymmetrical patterns of microbial nutrient limitation along the whole soil profile, and provide essential information in understanding nutrient limitations in deeper soils. These vertical and asymmetrical nutrient limitation patterns should be incorporated into future research studies priority.