The invention discloses a tobacco nitrate reductase NIA1 promoter and an expression vector and application thereof. The nucleotide sequence of the NIA1 promoter is the sequence shown in SEQ ID NO.1, or the key element which is highly similar to the sequence of a nitrate signal response element, wherein the NIA1 expression of the key element is removed or doubly regulated on the basis of retainingthe core promoter sequence of the sequence shown in the SEQ ID NO.1, or the nucleic acid sequence with equivalent functions derived from the sequence shown in the SEQ ID NO.1. The eukaryotic expression vector with the NIA1 promoter sequence is used for plant genetic transformation. Engineering bacteria with the NIA1 promoter sequence used for the plant genetic transformation is provided. The application of the NIA1 promoter in the initiation of a target gene expression is disclosed, and the target gene is a GUS gene. According to the tobacco nitrate reductase NIA1 promoter and the expression vector and application thereof, a foundation for verification of the NIA1 promoter nitric acid signal response function research is laid, and the tobacco nitrate reductase NIA1 promoter and the expression vector have broad prospects in improving nitrogen efficiency of plants, saving nitrogen fertilizer using amount, and developing resource-saving and environmentally-friendly agriculture.