A novel liquid crystal (LC) biosensor based on the recombination of split aptamer chip was developed for the detection of adenosine triphosphate (ATP). One glass slide of the LC cell is first modified with the TEA/DMOAP mixed self-assembled monolayer (SAM) to ensure the homeotropic alignment of LC molecules and the black background of LC cell optical image under the crossed polarized light. The single-strand ATP aptamer is split into two fragments in this method. One of which, as capture probe, is covalently immobilized on the SAM, while another is used as the detection probe. In the presence of ATP, those two fragments will be combined with each other and exhibit the same role as the ATP aptamer. This binding event leads to a great enhancement in the optical signal of the LC biosensor due to the space size from small to big, which can effectively disrupt the orientational arrangement of LCs, resulting in the corresponding changes of optical images under the crossed polarized light. But in the absence of ATP, those two fragments can not be combined with each other, and as a result, there is no orientational response of LCs and the optical image under the crossed polarized light is still black. So the LC-based imaging method has a sensitive and clear distinction between positive and negative results. On the basis of such an inhibition mechanism the LC biosensor can be used as an effective way to realize the detection of ATP. When the concentration of analyte ATP below a critical value (10 nmol/L), there is no clear change in the optical image. The results showed that the detection limit of ATP is 10 nmol/L. This study provides a simple and sensitive ATP LC biosensing approach and offers effective strategies for the development of small molecules LC biosensors.