Herein, we have meticulously derived the nanosized fluorescent aggregates from pyrene Schiff base (PS) in DMSO:water (10:90) ratio. The aggregation property of PS molecule was characterized by SEM and TEM measurements, revealed the aggregated particles are in spherical shape with ~3 nm in size. Moreover, aggregates exhibit a high fluorescence quantum yield (48%) which was effectively used for the in vitro bioimaging of two different cancer cells such as A549 and MCF-7 cells in which it exhibiting excellent biocompatibility. Further, it was estimated the capability of twofold acridine orange/ethidium bromide (AO/EB) staining to identify the apoptotic associated changes in cancer cells. Additionally, the aggregates were successfully demonstrated as a luminescent probe for the perceptive biomolecule detection of bilirubin. On the other hand, the PS molecule was successfully utilized for protein binding and metal ion sensing studies. The interaction of bovine serum albumin (BSA) with PS molecule in DMSO was using fluorescence spectroscopic method and nature of interaction was also confirmed through molecular docking analysis. The PS molecule also acts as an excellent sensor for biologically important Fe3+ ion with detection limit of 336 nM. Overall, PS molecule can be a prospective material in biological field both in solution as well as aggregated forms.