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We report perylenediimide-benzimidazolium based fluorescent ‘turn-on’ probe BIM-PDI for selective detection of human serum albumin (HSA) and bovine serum albumin (BSA) proteins. In HEPES buffer (0.1% DMSO), BIM-PDI self-assembles into aggregates and shows absorption maxima at 500 nm and weak fluorescence centered at 577 nm. The addition of HSA or BSA (1 × 10 −9 –5 × 10 −8 M) to the solution of BIM-PDI results in decrease in the emission intensity at 577 nm. However, further increase in concentration of HSA/BSA results in appearance of new blue shifted emission band at 540 nm. The minimum detection limit for HSA/BSA is 3.01 × 10 −10 M at 577 nm and 4.2 × 10 −8 M at 540 nm. On addition of BSA to the solution of BIM-PDI , the size of the aggregates decreased from 100 to 250 nm to <10 nm assigned to microencapsulation driven disassembly of BIM-PDI aggregates responsible for ‘turn-on’ response in fluorescence spectrum. Site-selective experiments using warfarin and diazepam drugs show that BIM-PDI preferably binds at site-I of HSA/BSA proteins. In clinical application of BIM-PDI , we estimated HSA content in blood serum and urine samples. BIM-PDI showed potential application in serum albumin protein tracking and imaging. [ABSTRACT FROM AUTHOR]