Since Terminal Protection of Small Molecule-Linked DNA (abbreviated as TPSMLD) was first found, TPSMLD-based assays have speedily prospered in small molecule-protein interaction study and sensing. However, most of the TPSMLD-based sensing strategies have adopted nuclease-assisted signal amplification, mainly by exo-nucleases, which adversely affect triggering the downstream reactions. Here we describe a simple, sensitive, and selective strategy for small molecule measurement taking biotin as a model. Due to the extension of the terminal protection mechanism to the polymerase and the introduction of the competition assay, a turn-on and expo-nential amplification signal was conveniently achieved. Benefiting from the high selectivity of TPSMLD and the exponential signal amplification assisted by terminal deoxynucleotidyl transferase (TdT)-endonuclease IV (Endo IV), biotin could be sensitively detected with a detection limit of 0.21 nM. Additionally, the recovery test was also executed in 1% spiked human serum with a satisfying recovery in the range of 95.8???98.0%, which demonstrated the robust anti-interference ability and potential clinical application of our proposed strategy. Predictably, by changing the small molecule-protein pair, this work could serve as a universal small molecule detection platform.