The determination of k and k values through kinetic analysis is crucial for understanding the intricacies of aptamer-target binding interactions. By employing kinetic ITC, we systematically analyzed a range of ITC data of various aptamers. Upon plotting their k and k values as a function of their K values, a notable trend emerged. Across a range of K values spanning from 28 nM to 864 μM, the k value decreased from 2×10 M s to 96 M s, whereas the k value increased from 1.03×10 s to 0.012 s. Thus, both k and k contributed to the change of K in the same direction, although the range of k change was larger. Since experiments are often run at close to the K value, this concentration effect also played an important role in the observed binding kinetics. The effect of these kinetic parameters on two common sensing mechanisms, including aptamer beacons and strand-displacement assays, are discussed. This work has provided the kinetic values of small molecule binding aptamers and offered insights into aptamer-based biosensors.