Although much effort has been made for studies on aptamer–target interactions due to promising applications of aptamers in biomedical and analytical fields, measurement of the aptamer–target binding constant and binding site still remains challenging. Herein, we report a sensitive label-free chemiluminescence (CL) strategy to determine the target concentration and, more importantly, to measure the target–aptamer binding constant and binding site. This approach is suitable for multiple types of targets, including small molecules, peptides, and proteins that can enhance the CL initiated by N-(aminobutyl)-N-ethylisoluminol functionalized gold colloids, making the present method a general platform to investigate aptamer–target interactions. This approach can achieve extremely high sensitivity with nanogram samples for measuring the target–aptamer binding constant. And the measurement could be rapidly performed using a simple and low-cost CL system. It provides an effective tool for studying the binding of biologically important molecules to nucleic acids and the selection of aptamers. Besides, we have also discovered that the 14-mer aptamer fragment itself split from the ATP-binding aptamer could selectively capture ATP. The binding constant, site, and conformation between ATP and the 14-mer aptamer fragment were obtained using such a novel CL strategy and molecular dynamic simulation.