A novel Label-Free Fluorometric Assay based on the recombination of split aptamer chip was developed for thedetection of adenosine triphosphate (ATP). In this strategy, the split aptamer was selected as a specific capture probe for the split two fragments aptamers could specifically form a ternary assembly in the presence of ligand and the two separate oligonucleotides lack secondary structures, thus not yielding false-positive or nonspecific signals, while the Thiazole orange(TO), an almost non-fluorescence dye in buffer solution, was used as signal probe, and the single-walled carbon nanotubes (SWCNTs) was applied to reduce the background signals. In the pH 80 Tris-HCl buffer solution, those two split aptamer fragments will be combined with each other to form a stable “aptamer-ATP-aptamer” composite structure upon interacting with its target ATP. The “sandwich” structure can’t wrap the sidewalls of the SWCNTs and is freed in solution, and TO shows agreat fluorescence enhancement when binding to the “aptamer-ATP-aptamer” composite structure. In the absence of ATP, the split aptamers, existing in a single-stranded state, bind to the surface of the SWCNTs via a π—π-conjugate interaction, and TO shows weak fluorescence because “sandwich”structure is not formed. In the system, the higher the ATP concentration is, the more the “aptamer-ATP-aptamer” sandwich recognition structure complex obtained, sois the fluorescence. Under the optimized experimental conditions, the ATP concentration in the range from 90×10-9 mol·L-1 to 10×10-7 mol·L-1 was linear with the ΔF/F0 value at the maximum fluorescence emission wavelength of 550 nm, r=0996 4, with a low detection limit of 267×10-9 mol·L-1. The recoveries of the method were 952%~104%, and the relative standard deviation (RSD) was 102%~454%, respectively. Based on the specific molecular recognition and high affinity of twosplit aptamers, the reaction product was shown that a “turn-on” fluorescence response to ATP with good selectivity, only a slight fluorescence change could be observed by GTP, CTP, and UTP (at a 200-fold higher concentration than that of ATP), indicating that UTP, CTP, and GTP could not interact with P1 and P1 to initiate the reaction. The method is simple, rapid, free-label, sensitive and accurate, and can be used for the determination of ATP in serum samples. Therefore, the present strategy has a great potential application prospect in the field of rapid detection of small molecular substances.