Currently, limited information is available on the enzymatic activities in ancient paleosols and permafrost, which are recognized as unique depositories of extant microorganisms. In this work, samples from buried and modern top Kastanozems (Haplic Calcisol), modern tundra (Gleysol) soils, and permafrost were tested for amylase activity based on hydrolysis of starch to low-molecular sugars that are important energy sources for microorganisms. Our study demonstrated the preservation of amylase in buried paleosols (300 and 2000 years old) and permafrost (15,000–60,000 years old). The enzyme activity in buried soils was 5–7 times lower than in top soils and did not significantly (P > 0.05) differ from permafrost and tundra soils. Exogenously added alkylresorcinols, valine, and glycine influenced amylolytic activity in soil and permafrost samples in the dose-dependent manner. The amylase response to stimulating concentrations of alkylresorcinols and amino acids was greater in buried paleosols (4.3–11.5-fold) than in top soils (1.9–3.8-fold) over the enzymatic activities in the controls. Positive response of soil amylase even to little changes in concentrations of the low-molecular-weight compounds may be important during recovery of biological activity of microorganisms. Results of this study contribute to a more complete evaluation of hidden biological potential of ancient soils and permafrost and are relevant to paleomicrobiology and soil biology.