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Abstract Extracellular enzymes play a key role in microbe‐mediated organic matter decomposition in soils, and the efficiency of these enzymes in substrate decomposition depends on their mobility and specific activity in soils. In this work, we explored the influence of biochar adsorption on extracellular enzyme activity across a spectrum of environmental conditions, from simple to complex. Batch adsorption results showed that biochar adsorption of two hydrolytic enzymes—α‐amylase and amyloglucosidase (AMG)—similarly decreases with pH and follows the Langmuir isotherm, suggesting electrostatic interaction between them. Activity of AMG and alkaline phosphatase (ALP), which belong to carbon and phosphorus cycling enzymes, was measured using a novel calorimetric method. The technique demonstrated advantages over conventional enzyme assays, such as in situ real‐time measurement of reaction rate and the ability to identify potential interferences. The technique enabled the measurement of specific activity of biochar‐adsorbed AMG, which ranged from 10% to 90% of that of free AMG. The effect of substrate adsorption on activity measurement was demonstrated through the examination of two substrates for ALP, which suggested the use of effective substrate concentration (instead of nominal concentration) in calculating enzyme activity kinetics. Soil column experiments showed that biochar amendment can affect the activity of AMG in starch hydrolysis through changing the mobility of AMG (and accessibility to substrate) and its specific activity. Results from this work improve our understanding of the effects of biochar adsorption on enzyme activity and suggest the need to appropriately interpret enzyme activity data and account for confounding processes.