Single-wavelength based fluorescence sensing scheme of ATP have been amply reported in literature whereas, only limited reports exist, where a ratiometric detection scheme for ATP is developed that attain high discrimination against a closely related competitive analyte pyrophosphate (PP_i), and constructed fromall commercially available molecules. In this work, we report a dye-polyelectrolyte supramolecular assembly which works as an efficient sensor platform for selective and sensitive detection of ATP, and provides a ratiometric output for ATP with LOD of 0.1 μM. The dye-polyelectrolyte supramolecular assembly consists of themonomer-aggregate equilibriumof a tetranionic dye, 1,3,6,8-pyrenetetrasulfonic acid (PTS) on the surface of a positively charged polyelectrolyte, poly(allylaminehydrochloride), PAH. This monomer-aggregate equilibrium is significantly modulated by ATP yielding an advantageous fluorescence ratiometric response. Importantly, the present dye-polyelectrolyte assembly shows very high selectivity for ATP over pyrophosphate which is seldom reported in the literature. More importantly, the sensor platformalso displays good response in human serum matrix, indicating its applicability in real-life scenarios. The sensor scheme involves all commercially available components which provide freedom from heavy dependence on synthetic molecules that involves laborious and time-consuming synthetic steps which are expected to significantly impact the prospect of this new sensor scheme in real-life applications.