Abstract: In order to identify miRNAs involving nicotine biosynthesis in tobacco, whole-genome miRNA identification was conducted and nicotine related miRNAs were predicted by bioinformatics. The functional verification of miRNA overexpressed plants was performed by qPCR and GC-MS technologies. The results showed that from 39 samples of cv. Honghuadajinyuan, including 5 tissues (root, stem, leaf, flower and axillary bud), at 3 developmental stages, a total of 2 925 potential miRNAs were identified, and 1 245 of which were conserved among other plants, and 43 candidates had already been reported in tobacco by other studies. The expression of NtQPT2 decreased by 48%-67% in miRNA2635 overexpressed plants, the expression of NtMATE1 decreased by 98% in miRNA203 overexpressed plants, and the average nicotine contents of two types of overexpressed plants decreased by about 69% and 62% respectively; which indicated that overexpressions of miRNA2635 and miRNA203 could significantly decrease the accumulation of nicotine by downregulating the expressions of NtQPT2 and NtMATE1.