2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene, also known as fluorescent brightener 0B, is used as a mixing raw material for plastics, coatings, road marking paints, etc., to achieve a whitening effect. In the prior art, the preparation method is to add a mixed solution of trichlorobenzene and toluene into a reaction kettle, then add 0B acid (i.e. thiophene 2,5-dicarboxylic acid), 0B phenol (i.e. p-tert-butyl-o-aminophenol) and boric acid, heat to about 120°C for condensation reaction, evaporate the water generated by the reaction together with the toluene, release the water with a water separator, and return the toluene to the reaction kettle. When the temperature of the reaction material rises to above 180°C, the toluene is evaporated, and the temperature continues to rise for reaction. The water is continuously separated by utilizing the immiscibility of trichlorobenzene and water until the reaction temperature reaches about 230°C. Based on the dehydration phenomenon, the reaction is judged to be complete, and then the material is cooled to below 30°C. The product is crystallized in trichlorobenzene, solid-liquid separation is performed, the trichlorobenzene mother liquor is used for recovery, and the obtained solid material is refined with xylene to obtain 2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene.