In this work, we found that the extract of leaves of a famous Traditional Chinese Medicine TiepiShihu (Dendrobium officinale Kimura et Migo) contained high content of flavonoids ranging from 7.66 mg·g−1 to 9.50 mg·g−1 dried weight and possed potent antioxidant activities. There was a strong positive correlation between DPPH radical scavenging rate and total flavonoids content. Furthermore, the antioxidant principles were determined by combination use of DPPH/ABTS+-HPLC assays and HPLC-DAD-based pattern recognition technologies. Their structure was further identified by on-line and off-line HPLC-ESI-MS/MS and following targeted isolation and NMR recognition. 14 phenolic herber-markers including quercetin-type flavonols and apigenin-type flavones were characterized from the leaves of D. officinale. The major flavonoid component of leaves was rutin and its content ranged from 1.33 to 2.89 mg·g−1 dry weight. In summary, whole results indicated that the leaves of D. officinale might be developed as antioxidant sources for medicinal and health food resources.