The combinatorial fluorescent dapoxyl dye library was prepared by both solution- and solid-phase synthesis, generating 80 unique dapoxyl derivatives. A fluorescence-based screening toward human serum albumin (HSA) found one highly sensitive HSA binder ( A41-S) with over 55-fold intensity change. Displacement assay showed the selective binding of A41-S to the site I of HSA, addressing its potential to be a highly selective and sensitive HSA probe.