Abstract: In order to find an effective way to extract RNA in tobacco leaves and provide theoretical basis for tobacco molecular biology research, tobacco leaves were used as material, 3 methods including RNA isolation kit, Trizol method and RNAiso method were used to extract RNA, then RNA were detected by gel electrophoresis and spectrometer. The results indicated that the operation of RNA isolation kit was very easy, but the extraction was the least; Trizol method was convenient and efficient, but there were proteins and a little degradation in RNA; in contrast, the concentration of RNA extracted by RNAiso method was very high and ranged from 100 to 500 ng/μL, A₂₆₀/A₂₈₀ was from 1.8 to 2.0, RNA bands were clear and bright without degradation, and the brightness of 28S band was about twice that of 18S band; RT-PCR result showed that 2500 bp target gene consistent with the reference sequence was obtained, and it could be used in functional verification. Hence, RNAiso method was the best way to extract total RNA in tobacco leaves and had been used in many laboratories.