The invention provides a method for preparing 2,5-disubstituted thiophene, wherein the method comprises the following steps: carrying out a sulfur addition cyclization reaction of sodium sulfide and 1,4-disubstituted-1,3-butadiyne under a condition of an organic solvent, to obtain 2,5-disubstituted thiophene. According to the preparation process, the preparation method provided by the invention has no addition of any catalyst containing metal ions, and even has no addition of any type of catalyst, so that any impurity substances or ions cannot be introduced in the reaction process. In addition, the purity of 2,5-disubstituted thiophene obtained by the preparation method provided by the invention is more than 99%, and the yield is more than 85%.