A electrochemical biosensor based on an aptamer/nano silver probe and an EXO I enzyme. Nano silver nanoparticles having the functions of identifying a target object and generating electrochemical signals and modified by an aptamer are used as a biological probe for detecting target biomolecules, under the initiation of the target object and the assistance of a complementary probe and the EXO I enzyme cyclic shear amplification, and by means of the DNA complementary pairing principle, the probe can be gathered on the surface of a gold electrode, the larger the concentration of the target biomolecules is, the larger the gathering degree of the induced aptamer/nano silver probe is, and meanwhile an EXO I exonuclease target cyclic amplification strategy is introduced in the identification process of the target object, so that the electrochemical biosensor can sensitively and efficiently detect target biological substances.