The invention relates to a tobacco cytochrome C gene NtCYP94B3s and application thereof in increasing of the jasmonic acid content of tobacco. The nucleotide sequence of the tobacco cytochrome C geneNtCYP94B3s is shown as SEQ ID NO:1 in a sequence table, or a nucleotide sequence hybridized with a DNA sequence defined by SEQ ID No:1 in the sequence table, or a DNA sequence which has more than 70%of homology with the DNA sequence defined by SEQ ID No:1 in the sequence table and encodes same functional protein. In the invention, the expression of the tobacco endogenous gene NtCYP94B3s is inhibited in tobacco plants, so that the contents of JA and JA-IIe in tobacco leaves are obviously increased; and the tobacco cytochrome C gene NtCYP94B3s has a wide application prospect in the field of high-JA and high-JA-IIe-content breeding of plants.