This study developed an efficient and reliable system for inducing polyploidy in Anoectochilus formosanus Hayata, a top-grade medicinal orchid. The resulting tetraploid gave a significant enhancement on various agronomic traits, including dry weight, fresh weight, shoot length, root length, leaf width, the size of stoma, and number of chloroplasts per stoma. A reduction of the ratio of length to width was observed in stomata and leaves of the tetraploid, and consequently, an alteration of organ shape was found. The major bioactive compounds, total flavonoid and gastrodin, were determined by the aluminum chloride colorimetric method and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), respectively. The tetraploid produced significantly higher contents of total flavonoid and gastrodin in the leaf, the stem, and the whole plant when compared with the diploid. The resulting tetraploids in this study are proposed to be suitable raw materials in the pharmaceutical industry for enhancing productivity and reducing cost.