The invention discloses an extracting purifying and detection method of neophytadiene in tobacco, and applications thereof. The extracting purifying and detection method comprises following steps: analytically pure dichloromethane is added into a tobacco sample, ultrasonic extraction is carried out, and filter paper filtering is carried out; a certain amount of an obtained crude extraction solution is delivered through a silica gel column for preliminary separation purification; an obtained product is eluted with petroleum ether, and an obtained eluent is concentrated, and is subjected to purifying and concentration via preparative chromatography; and back-extraction with chromatographically pure dichloromethane is carried out so as to obtain a neophytadiene sample. According to the extracting purifying and detection method, cured tobacco leaves are taken as a raw material for direct extraction; the raw material is easily available, and cheap; compared with chemical synthesis, the extracting purifying and detection method possesses following advantages: a few steps are needed, operation is simple, it is found by analysis and detection that the purity of the prepared neophytadiene is 99.42% or higher, and is much higher than that of commercially available neophytadiene, the finished product contains no neophytadiene dipolymer, and accuracy is higher when the finished product is taken as a standard substance in quantitative analysis of tobacco aroma substances and precursors of the tobacco aroma substances.