The invention discloses a method for detecting residual kanamycin and application thereof, and belongs to the technical field of analysis chemistry, wherein the detection method is based on aptamer initiated cyclic enzyme digestion of exonuclease III (Exo III). According the method, mercapto self-assembly is utilized, single stranded DNA (ssDNA) that is complementary with kanamycin aptamer (K-aptamer) is grafted on the surface of a gold electrode, then a little amount of K-aptamer is added, and the K-aptamer is matched with ssDNA to form a little amount of double stranded DNA (dsDNA). Then Exo III is added, the ssDNA in dsDNA can be specifically digested by Exo III, thus the residual aptamers are released, the released aptamers can combine with ssDNA to form dsDNA, another enzyme digestion is triggered then, and after several circulations, the modified ssDNA is completely digested by enzymes. Kanamycin exists in the system, through the combination between kanamycin and aptamers, the circulation enzyme digestion is inhibited, thus ssDNA is preserved, and the amount of preserved ssDNA is related with the concentration of kanamycin. An electro-analysis method is used to absorb electrical signal molecules namely hexaammine ruthenium (RuHeX) through static electricity absorption so as to quantitatively detect residual kanamycin; and the method has high sensitivity and can sensitively detect residual kanamycin in milk.