Abstract: Objective Comprehensive analyze the microscopical character and Infrared Spectroscopy(IR) character of Dendrobium officinale, D. moniliforme and D. devonianum in order to provide the basis for distinguishing the three medicinal Dendrobii. Methods The freehand section and micrometer analysis were conducted to study the authentication chracters of the stems of three medicinal Dendrobii. FT-IR as well as the second order derivative FT-IR to analyze the IR characters of the three medicinal Dendrobii. Results The thickness of cuticula of D. officinale were 4.9μm that was fairly thiner than the other two species. The thickness scale for cell of stem epidermis in D. officinale is 9.1μm belong to non uniform thickness, D. moniliforme has non thickness, and D. devonianum has uniform thickness. The special peak in the second order derivative for D. moniliforme is 1585、1217 and 1049cm-1, D. officinale and D. devonianum has the same special peak of 1569、1450、1225 and 1073 cm-1, D. moniliforme and D. devonianum has the same special peak of 1011 cm-1. Conclusions Comprehensive application for the method of microscopical and IR identification can precisely distinguish the three medicinal Dendrobii.