Thrombin has a strong correlation to a variety of diseases, and it is of great significance to improve the sensitivity of thrombin detection. Herein, we report a biosensor for highly sensitive thrombin detection (17 fM) by combination of click chemistry and surface-initiated atom transfer radical polymerization (SI-ATRP). A sandwich structure is formed among thiol-modified aptamer-1, thrombin and azido-labeled aptamer-2. Then the initiators of SI-ATRP (propargyl alpha-bromoisobutyrate, PBiB) can be introduced to electrode surface via electrochemically mediated Cu(I)-catalyzed azide-alkyne cycloaddition (eCuAAC). Subsequently, SI-ATRP is activated based on the reaction that ascorbic acid reduced Mn(III)meso-tetra(4-carboxyphenyl)porphine (Mn(III)-TCPP) to Mn(II)meso-tetra(4-carboxyphenyl)porphine (Mn(II)-TCPP), and numerous monomers are grafted to electrode surface via SI-ATRP. The limit of detection is enhanced about 4.9 x 10(3)-fold compared with previous strategy, the sensitivity of this biosensor can be improved and this method also have desirable stability and reproducibility. (c) 2019 The Electrochemical Society.