Amongst the various existing methods of analyte quantification, fluorescent-based methods, especially the ratiometric methods, continue to gain significant attention due to their high reproducibility, low environmental influence, and self-calibrating behavior. This paper presents the modulation in a monomer-aggregate equilibrium of coumarin-7 (C7) dye at pH ∼ 3, under the influence of a multi-anionic polymer, poly(styrene sulfonate) (PSS), leading to a significant modification in the ratiometric optical signal of the dye. At pH ∼ 3, cationic C7 formed aggregates in the presence of PSS via a strong electrostatic interaction, resulting in the development of a new emission peak at 650 nm at the expense of the monomer emission at 513 nm. Such contradicting changes in fluorescence intensities at two different wavelengths gave rise to a ratiometric signal, which was found to be highly sensitive towards external stimuli such as pH, and ionic strength. The stability of the C7-PSS complex was found to decrease as the pH of the solution was increased beyond 5, which indicated the decline in the electrostatic attraction between C7 and PSS due to the deprotonation of the C7 dye. Furthermore, an increase in the monomeric peak and a concomitant decrease of the aggregate peak with added salt in the solution (at pH ∼ 3) clearly justified the presence of an electrostatic attraction between C7 and PSS for the complex formation. This was further validated by the excited-state lifetime measurement of the C7-PSS complex, which showed a systematic increase in lifetime contribution from the monomeric species at the expense of aggregated species, as the concentration of NaCl increased in the solution. Thus, protamine (Pr), being a highly positively charged polypeptide, largely affected the monomer-aggregate equilibrium of the C7-PSS system, leading to a phenomenal change in the ratiometric signal, which was utilized to quantify with LOD as low as ∼2.8 nM in buffer for the bio-analyte Pr. Moreover, the ratiometric response of the C7-PSS assembly demonstrated excellent selectivity towards Pr, facilitating its practical relevance for the quantification of Pr in a 1% human serum matrix. Therefore, the studied C7-PSS can be utilized as a potential candidate for the quantification of the protamine even in complex biological media.