Two levels of nucleic acids-based isothermal amplification normally require a long reaction time due to the low concentration of catalyst, which limits its practical application. A sensitive fluorescence assay of chloramphenicol (CAP) was developed coupled with two-level isothermal amplification using a self-powered catalyzed hairpin assembly (CHA) and entropy-driven circuit (EDC). CAP can bind with its aptamer to open its closed structure. The opened hairpin can initiate self-powered CHA and EDC. The product of CHA can circularly catalyze the CHA with increasing concentration. In principle, the product of CHA plays the role of catalyst and increases with the progression of the reaction. Compared with the normal two levels of amplification, the amplification efficiency of our strategy is much higher due to the self-powered reaction by the CHA product. Thus, the reaction time is shortened to 110 min in this strategy. Moreover, the detection limit for CAP can achieve 0.1 pM and shows promising prospects for practical application.