Cyclic diadenylate monophosphate (c-di-AMP) and P-1,P-5-diadenosine-5' pentaphosphate (Ap5A) have been determined to play important roles in bacterial physiological processes and human metabolism, respectively. However, few, if any, methods have been developed that use fluorescent sensors to sense c-di-AMP and Ap5A in the real world. To address this challenge, this study presents a fast, convenient, selective, and sensitive assay for quantifying c-di-AMP and Ap5A fluorescence based on the competitive binding of diadenosine nucleotides and a polyadenosine probe to coralyne. The designed probe consists of a 20-mer adenosine base (A(20)), a fluorophore unit at the 5'-end, and a quencher unit at the 3'-end. Through A(2)-coralyne-A(2) coordination, coralyne causes a change in the conformation of A(20) from that of a random coil to a folded structure, thus enabling the fluorophore to be close to the quencher. As a result, fluorescence quenching occurs between the two organic dyes. When the A(20).coralyne probe encounters the diadenosine nucleotide, the resulting complex of coralyne and diadenosine nucleotides forces the removal of coralyne from the probe. Such a conformational change in the probe leads to the restoration of fluorescence. Within a short analysis time of 1 min, the proposed probe provides high selectivity toward c-di-AMP and Ap5A over other common nucleotides. The probe's detection limit at a signal-to-noise ratio of 3 for both c-di-AMP and Ap5A were estimated to be 0.4 and 4 mu M, respectively. The practicality of the proposed probe was demonstrated by quantifying c-di-AMP in bacteria lysates and Ap5A in human tears.