Soil enzymes play a key role in soil organic matter dynamics in terrestrial ecosystems; however, the relationship between soil enzyme activity and the chemical composition of soil organic matter remains unclear. We analysed the soil organic matter (SOM) of five types of alpine grassland ecosystems in the Tibetan Plateau at the molecular level using pyrolysis gas chromatography/mass spectrometry (Py-GC/MS), which allowed us to examine the relationship between the patterns of enzyme activities and SOM composition. We found that the level of activity of carbon (C)-cycling enzymes (invertase, beta-1,4-glucosidase, polyphenol oxidase, and peroxidase), nitrogen (N)cycling enzymes (arylamidase), and phosphorus (P)-cycling enzyme (alkaline phosphatase) was significantly different among the five grassland ecosystems. In addition, we found significant variation among the different grassland ecosystems in terms of the activity of enzymes involved in C-cycling less so with those enzymes involved in N-cycling. In general, SOM was dominated by N-compounds (-32.47-51.76%), polysaccharides (-10.67-20.42%), and fatty acids (-7.03-20.33%) in the different alpine grasslands. The most abundant compounds in SOM were D-alanine (10.06-20.77%), 9-octadecenamide, (Z) (4.43-9.68%), and 13-docosenamide (1.52-14.76%). Correlation analysis showed that the N-compound abundance was negatively correlated with nitrate reductase. Polysaccharides were positively correlated with activity levels of invertase and cellulase. Fatty acids were positively correlated with polyphenol oxidase activity and negatively correlated with alkaline phosphatase activity. Some abundant compounds consistently showed strong correlations with activity levels of invertase and cellulase, including toluene, styrene, benzene, phenol, furfural, pyrrole, p-cresol, 2-methylphenylacetylene and indole (positive correlation), 13-docosenamide, dodecanamide and o-hydroxybiphenyl (negative correlation). Furthermore, Gram-positive and Gram-negative bacteria also play a significant role in regulating SOM-enzyme interactions. These findings confirmed the strong effects of SOM composition on the activity of invertase and cellulase, indicating that invertase and cellulase play an important role in regulating SOM to some extent, resulting in complex interactions among organic-enzyme-microbes.