In the present paper, a tri-step infrared (IR) spectroscopy was used to study Scutellaria baicalensis Georgi (SBG) harvested in spring and autumn. The positions of peaks in the IR spectra of SBG harvested in spring and autumn were rather similar. However, according to the differences in the relative intensities of those characteristic peaks which include the ester carbonyl CO absorption peak at 1 740 cm-1, the peak near 1 614 cm-1 assigned to the flavonoids, and the peak near 1 071 cm-1 assigned to the carbohydrates, the amount of flavonoids and esters of spring SBGs was higher than that of autumn SBGs. Their carbohydrates were different by comparing the second derivative infrared spectra in the range of 1 300～400 cm-1. Furthermore, the above differences were visually validated by two-dimensional correlation spectroscopy(2D-COS). Moreover, the FTIR spectra of 16 batches of SBG harvested in spring and autumn were analyzed with principal component analysis, and subsequently they were exactly identified and classified. Therefore, tri-step identification of IR spectroscopy combined with principal component analysis can be employed to fast and accurately identify the SBG harvested in spring and autumn and differentiate the differences of their chemical constituents.