The invention discloses a preparation method of a paper-based photocathode biosensor for detecting microRNA. A gold nano-particle layer is coated in a working area of a paper-based carbon working electrode; polyhedral cuprous oxide is electrodeposited and lots of silver sulfide quantum dots can be loaded, and a photoelectric sensitized structure is formed, so that the cathode photocurrent is increased substantially; and in the presence of the target microRNA, a double-strand-specific-nuclease-induced target cycle reaction and a cascaded stem-branch hybridization strand reaction are made to obtain a negatively charged dendritic DNA double-helix structure. After assembling of positively charged gold nanoparticles, a gold tree with the good glucose oxidase mimicking activity is formed, so that the glucose oxidation reaction is catalyzed, the dissolved oxygen of the electron acceptor is consumed, the cathode photocurrent signal is reduced, and sensitive detection of the microRNA is realized.