Spectra-structure interrelationship is still the weakness of NIR spectral assignment. In this paper, a comprehensive investigation from chemical structural property to natural chemical compounds was carried out for NIR spectral assignment. Surprisingly, we discovered that NIR absorption frequency of the skeleton structure with sp2 hybridization is higher than one with sp3 hybridization. Specifically, substituent was another vital factor to be explored, the first theory discovery demonstrated that the absorption intensity of methyl substituted benzene at 2330 nm has a linear relationship with the number of substituted methyl C-H. The greater the number of electrons given to the substituents, the larger the displacement distance of absorption bands is. In addition, the steric hindrance caused by the substituent could regularly reduce the intensity of NIR absorption bands. Furthermore, the characteristic bands and group attribution of 29 natural chemical compounds from 4 types have been systematic assigned. These meaningful discoveries provide guidance for NIR spectral assignment from chemical structural property to natural chemical compounds.