Abstract: AIM: To adopt a TLC fluorescent scanning method for the separation and determination of the alkaloids: hydroxyevodiamine (I), N-formyldihydrorutaecarpine (II), goshuyuamide-I (III), evodiamine (IV) and rutaecarpine (V) in Evodia rutaecarpa and its preparation. METHODS: Chloroform—petroleum ether—methanol—cyclohexane (5∶3∶0.4∶0.4) was used as developing solvent on the HPTLC silica gel plate for first development; and chloroform—methanol (4.1∶0.3) for the second development. After that, the spots were derivatized by concentrated hydrochloric acid and then determined with a Shimadzu CS-930 TLC scanner. RESULTS: The calibration curves were linear in the range of 20.5～245 ng for the five alkaloids and the recoveries were 96.8%～104.5%. The method has been applied to the analysis of various samples and their preparations. CONCLUSION: This highly sensitive method required small amount of sample. It offered a scientific basis for seeking and utilizing botanic resources, and controlling the quality of herb medicines and preparations.