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The application discloses an enzyme-electron medium micro-nano composite for biosensing and a preparation method and application thereof. A dehydrogenase for electrochemical detection and an electron medium are jointly loaded in a cavity of a carrier, the carrier is contracted through chemical or physical stimulation, and electron medium molecules in the cavity are forced to be distributed around active centers of the dehydrogenase, so that a contractible limited microsphere structure is obtained. The enzyme-electron medium micro-nano composite obtained by the application can significantly improve a response current generated by unit enzyme catalysis, enhance the stability of the enzyme, and effectively prolong the service life of a sensor.