Photometry was employed to study the optimum extraction conditions of anthraquinone derivatives from rhizomes of Rheum officinale Baill in this study. The influences of extraction solvents (chloroform, benzene, ethanol, methanol, and glycerol), acid, and extraction time on the extraction yield were discussed. The results indicate that, to the Rhubarb rhizomes powder with the average particle size 0.18 mm, the conditions of the extraction solvent composed by chloroform, glycerol, and sulfuric acid (20%) in the ratio of 4:1:1 (v : v), the weight of dried Rhubarb to the solvent volume in the ratio of 1:12 ew: vT, extraction time of 110 min, the anthraquinone derivatives extraction could achieve the best yield. And the antibacterial tests showed the raw extraction products had the MIC (minimal inhibitory concentration) of 20 μg=mL and 30 μg=mL to Staphylococcus aureus and Escherichia coli, respectively.