Enzymes in the “Auxiliary Activities” (AA) 3 family in the Carbohydrate-Active enZYmes Database (CAZy) belong to the glucose-methanol-choline family. All the members in AA3 family use flaxin-adenine dinucleotide (FAD) as a cofactor, assisting enzymes from other AA families to perform their functions via their reaction products (H₂O₂ or hydroquinone), or facilitating glycoside hydrolase to degrade lignocellulose. According to the structure and sequence similarity, the enzymes from AA3 family were further subdivided into 4 subfamilies, mainly including cellobiose dehydrogenase, aryl-alcohol oxidase and glucose oxidoreductase, alcohol oxidase, pyranose oxidoreductase. On account of the high expression level, efficient catalysis, diversity of biotransformation, and fast electron transport kinetics, AA3 enzymes have become an interesting target for electrochemical biosensors. In order to improve the performance parameters of biosensors, a tremendous development has been obtained in the field ranging from the production of new AA3 enzymes with tailor-designed properties, such as the improved specific activity, low O₂ sensitivity and enhanced redox mediator interaction, to their efficient immobilization in a variety of nanomatrices. Thus in this paper, we provide an overview of the phylogenetic, molecular, and catalytic properties of CAZy-AA3 family enzymes, and the latest research progress of AA3 enzymes used in electrochemical biosensors was also summarised. In the development trend of the future study, it calls for the combination of protein engineering skills with expertise on redox mediator/polymer synthesis and electrochemistry to facilitate the application of AA3 enzymes in biosensor.