Sensitive and selective recognition of Adenosine tri-phosphate (ATP) is considered very important due to its crucial roles in various metabolic pathways and biological processes. Most of the developed sensor platform for ATP suffers from poor solubility in the aqueous medium, respond via single wavelength based measurements or have complicated synthesis procedures for employed probes, which are some of the major disadvantages. Consequently, to address these limitations, herein, we report a ratiometric fluorescence sensor for the detection of ATP, on the basis of interaction between an anionic polyelectrolyte, poly(styrene-sulfonate) (PSS) and a molecular rotor based cationic probe, Thioflavin-T (ThT). The developed sensor platform operates by the fluorescence turn-off response of the fluorescent ThT-PSS assembly on the introduction of Cu²⁺ (disassembly) followed by the turn-on response on interaction with ATP (reassembly) that enables it to act as a sensing platform for the ratiometric identification of ATP. Overall, the developed sensor platform offers a simple, label-free, sensitive and highly selective recognition of ATP with LOD of 3.3 μM over the linear range of 0–260 μM in water. Importantly, the response of the developed sensor platform towards ATP in the serum sample with LOD of 10.1 μM indicates the potential of real-life utilization of this probe in near future.