The invention relates to a primer combination and a kit for identifying flue-cured tobacco Nanjiang NO.3, application and a detection method, and belongs to the technical field of tobacco variety identification. According to the primer combination and the kit for identifying the flue-cured tobacco Nanjiang NO.3, application and the detection method, a 420K Tobacco SNP array is utilized to conduct whole-genome SNP typing on the mainly-planted varieties of tobacco in our country in recent years, a set of specific SNP markers suitable for identifying the flue-cured tobacco Nanjiang NO.3 are obtained through screening according to intervarietal polymorphic SNP sites, wherein the number of the specific SNP markers is twelve; twelve SNP site flanking sequences obtained from a reference genome of cultivated tobacco are aligned, the sequences are as shown in SEQ ID NO: 1-12, and primers are designed based on the sequences; and the sites are subjected to typing detection by utilizing the designed primers and adopting a matrix-assisted laser desorption-ionization time of flight mass spectrometry technology. An SNP typing result is obtained according to a detection result, and whether a sample is the Nanjiang NO.3 or not is identified. By adopting the detection method, the needed detection sample amount is small in the detecting process, the detection throughput is high, and the identification result is accurate.