Pyrene-based cyclophanes have been synthesized with the aim to realize ab ellows-type sensing mechanism for the ratiometric detection of nucleotide concentrations in ab uffered aqueous solution. The sensing mechanism involves the encapsulationo fan ucleobase between two pyrene rings,w hich affects the monomer-excimer equilibrium of the receptori nt he excited state. The nature of the spacer and its connection pattern to pyrene rings have been varied to achieve high selectivity for ATP. The 1,8-substituted pyrene-basedc yclophane with the 2,2'-diaminodiethylamine spacer demonstrates the best selectivity for ATPs howing a 50-fold increase in the monomer-excimer emission ratio upon saturation with the nucleotide. The receptor can detect ATPw ithin the biological concentrations range over a wide pH range. NMR and spectroscopic studies have revealed the importance of hydrogen bonding and stacking interactions for achievingarequired receptors electivity.T he probe has been successfully applied for the real-time monitoring of creatine kinase activity.