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Abstract To study how the understory vegetation influences the catalytic properties of extracellular hydrolases, we established a paired treatment experiment, with understory vegetation and litter removed from one treatment (understory removal, UR) and litter removed and the understory vegetation left intact in the other (control, CK), in a subtropical Chinese fir (Cunninghamia lanceolata) plantation. We used fluorescent substrates to determine the maximum velocity (V max), the affinity of hydrolases to the substrates (K m), and the catalytic efficiency (V max /K m) of three extracellular hydrolases (β-1,4-glucosidase (βG), β-1,4-N-acetylglucosaminidase (NAG) and acid phosphatase (AP)). We found that the V max values for βG and NAG were 23.0% and 16.8% lower, respectively, in the UR treatment than in the CK treatment, and that the V max for AP was similar in both treatments. The K m and V max /K m for all the three hydrolases remained steady after the understory vegetation was removed. The soil C and N contents and the bacterial and fungal biomass were generally positively correlated with the V max values. Thus, understory vegetation had more influence on the activities (V max) than on the substrate affinities of extracellular hydrolases (K m). Microbes tended to concentrate on maintaining the catalytic efficiency when the SOC contents were between 15.8 and 20.3 g kg−1 when the understory vegetation was removed from a subtropical Chinese fir forest, and the catalytic efficiency could decrease under extremely low soil C contents. We suggest that understory vegetation should be maintained to sustain the potential microbial activity in subtropical Chinese fir forests. Highlights • The V max of βG and NAG, but not AP, decreased when understory vegetation was removed. • The K m and V max / K m values did not change when understory vegetation was removed. • Soil C and N and microbial biomass were mainly positively related to the V max. • Understory vegetation should be maintained to sustain potential microbial activity. [ABSTRACT FROM AUTHOR]