A novel compound biotinylated emodin was synthesized by a two-step acyl chloride method which connects the biotin to emodin with esterification reaction. The product was characterized with ultraviolet-visible spectrophotometry, fourier transform infrared and high-performance liquid chromatography tandem mass spectrometry techniques. Its antibacterial activity against Staphylococcus aureus CMCC 26003 was investigated, and the emodin- and biotinylated emodin-caused antibacterial mechanism was proposed. It was shown that the product was isolated and activity of emodin was remained. These results indicated that our study provides a kind of chemosynthesis method under mild conditions and a strong molecular tool for investigating the emodin-binding protein.