A fluorescent dye, 2,5-bis(6-amine-benzoxazol-2-yl)thiophene (BBTA), was synthesized by a two-step reaction from starting material 2,5-bis(benzoxazol-2-yl)thiophene (BBT). BBTA exhibited strong emission and large Stokes shift in solvent, and the largest Stokes shift (Dl ¼ 186 nm or Dn ¼ 8572 cm À1 ) was obtained in buffer solution (PBS, pH ¼ 7.2) with 5% (v/v) of polyethylene glycol monomethyl ether (mPEG 550 , MW ¼ 550) as additive. Application of BBTA to live cell imaging showed that BBTA was clearly expressed in mitochondria with high contrast. Besides, BBTA showed low cytotoxicity and excellent photo-stability.Scheme 2 Synthetic route for BBTA.Scheme 1 Chemical structure BBTA.This journal is