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Electrochemical enzyme-based biosensors are one of the largest and commercially successful groups of biosensors. Integration of nanomaterials in the biosensors results in significant improvement of biosensor sensitivity, limit of detection, stability, response rate and other analytical characteristics. Thus, new functional nanomaterials are key components of numerous biosensors. However, due to the great variety of available nanomaterials, they should be carefully selected according to the desired effects. The present review covers the recent applications of various types of nanomaterials in electrochemical enzyme-based biosensors for the detection of small biomolecules, environmental pollutants, food contaminants, and clinical biomarkers. Benefits and limitations of using nanomaterials for analytical purposes are discussed. Furthermore, we highlight specific properties of different nanomaterials, which are relevant to electrochemical biosensors. The review is structured according to the types of nanomaterials. We describe the application of inorganic nanomaterials, such as gold nanoparticles (AuNPs), platinum nanoparticles (PtNPs), silver nanoparticles (AgNPs), and palladium nanoparticles (PdNPs), zeolites, inorganic quantum dots, and organic nanomaterials, such as single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), carbon and graphene quantum dots, graphene, fullerenes, and calixarenes. Usage of composite nanomaterials is also presented.