The invention relates to the technical field of biosensor, in particular to a fluorescence biosensor based on amplification of a hybridization chain reaction. The problems that the specificity and sensitivity of the method for detecting the kanamycin is low, and the cost is high in the above prior art are solved. According to the fluorescence biosensor based on the amplification of the hybridization chain reaction, the cyclic amplification effect is achieved through the hybridization chain reaction between the endonuclease IV and the chain. The mixture is reacted homogeneously by fluorescenceresonance energy transfer of the fluorophores and the quenching group. The preparation method comprises the following steps of: preparing gold nanoparticles; modifying Walker and Track to the surfaceof gold nanoparticles; mixing the labeled gold nano solution with the homogeneous reaction solution; performing hybridization chain reaction, and detecting the fluorescence; using the specific recognition of the nucleic acid Aptamer, using the nucleic acid Aptamer for high specific detection of the target kanamycin; and using the amplification of the hybridization chain reaction to achieve signalamplification.