Analyzing the chemical components of traditional Chinese medicines containing multiple isomers is thorny. Here, an analytical strategy by using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry coupled ion mobility spectrometry was applied to characterize the main compounds from the fruits of Tribulus terrestris, which significantly boosted the separation efficiency and broadened the capacity of chromatographic column. A total of 155 chemicals including 120 steroidal saponins, 13 flavonoids, 20 alkaloids and 2 ferulic acids were identified or tentatively identified using the propounded method. An α-glucosidase inhibition test was conducted to compare whether the aptness of Tribulus terrestris differed from different origins. The results indicated that the activity of Tribulus terrestris from Inner Mongolia was better than that from Jilin and Hebei. Principal component analysis was next employed to seek out the potential chemical markers among these Tribulus terrestris from three origins. There were 13 substances exist significant differences in content of Tribulus terrestris from the three producing areas. These significant differences involve 11 steroidal saponins and 2 alkaloids. Among them, Inner Mongolia possessed the highest contention of all the 11 saponins. This suggested steroidal saponins may emerge huge potential of α-glucosidase inhibition activity. In conclusion, the present study furnished the identification of chemical components of medical herbs with various isomers as well as disclosed the latent diabetes treatment potential of steroidal saponins in Tribulus terrestris.