In recent years exploration of aggregation induced emission from the naturally occurring luminogens has garnered significant attention across the globe due to their widespread applications in diverse fields of modernday research. Herein, we delineate multi-anionic polymer, polystyrene sulfonate (PSS) templated aggregation induced emission (AIE) of a medically important and naturally origin alkaloid, berberine (BBR) and its application towards the quantification of an important bio-analyte, protamine (Pr). Cationic BBR at the vicinity of the polyanionic PSS, partially loses its positive charge which causes the reduction in the electrostatic repulsion between the adjacent dye molecules, promoting their aggregation via π-π staking over the polymer surface. This substantially changes the microenvironment of the dye surrounding, leading to the lowering in its structural swiftness and tendency of intramolecular charge transfer (ICT) state formation. Thus, the dye exhibits amplified emission in the aggregated state. Increase of average lifetime and sluggish nature of anisotropy decay of BBR in the presence of PSS support structural confinement and weakening of non-radiative deexcitation pathways for the dye through the formation of BBR-PSS supramolecular assembly. The formed BBR-PSS assembly displays high sensitivity towards external parameters like ionic strength, temperature but unresponsive to a wide range of pH. Importantly, BBR-PSS has been found to be highly sensitive and selective towards an important polypeptide, Pr. Owing to its high positive charge density, Pr disintegrates the BBR-PSS complex, producing fluorescence turn-off response for the BBR-PSS complex which enables us to quantify Pr in aqueous solution. Notably, this AIE based complex is quite efficient to detect protamine even in 1% human serum matrix as low as ~ 89 nM in spite of presence of various other interfering bioanalytes.