An amphiphilic fluorescent probe, 3‐dodecylamino dihydrogen imidazo[2,1‐α]benz[de]isoquinolin‐7‐one (compound 3), was used to sense the aggregate formation process of bovine serum albumine (BSA), sodium dodecyl sulfate (SDS) and their mixed system. The fluorescence intensity of 3 was significantly affected by the adding order of SDS and BSA, and SDS can be distinguished from other surfactants with the aid of BSA, but only when 3 is allowed to interact with BSA first. The results revealed that compound 3 is preferentially sited in the hydrophobic region of BSA, and thermodynamically in SDS‐BSA mixed aggregate. Sodium phosphate buffer solution (PBS) and BSA played important but distinct roles in distinguishing SDS micelle from the others.