The invention relates to a kit and a method for regulating seed germination and the application of the kit and belongs to the technical field of genetic engineering. The kit for regulating seed germination comprises: a knockout vector of a procyanidine substance regulation factor NtMYB330 gene; the method for regulating seed germination is characterized in that a procyanidine substance regulation factor NtMYB330 gene in seeds is knocked out. The amino acid sequence of the procyanidine substance regulatory factor NtMYB330 is as shown in SEQ ID NO. 2. The germination rate of the NtMYB330 gene knockout mutant seeds obtained by adopting the kit or the method disclosed by the invention is averagely improved by 87.95%. Therefore, the tobacco NtMYB330 gene mutation has a relatively great application prospect in the aspect of improving the germination rate of the tobacco seeds.