Aim To establish an in vitro fluorescence spectrophotometry based on the end-product malondialdehyde(MDA)for evaluating hydroxyl radical-scavenging ability, and compare the advantages and disadvantages of fluorescence and visible methods.Methods The reaction time, temperature, and the concentration of key reactant deoxyribose were investigated and optimized respectively.Under different solvent conditions, sensitivity and the measurement window of two methods were compared.The hydroxyl radical scavenging ability of tanshinone I was determined by these two methods.Results The optimal temperature and time was 37 °C and 60 min, and the concentration of deoxyribose was 2.8 mmol·L-1.The limit of detection for the fluorescence method(4.49 nmol·L-1)was much lower than that of the visible spectrophotometry(39.15 nmol·L-1).The ratio of model/control(the measurement window)of the fluorescence method was much larger than that of visible spectrophotometry in both the aqueous system and the organic system(containing DMSO).Within the concentrations of 62.5 mg·L-1-1 000 mg·L-1, tanshinone I showed scavenging ability on hydroxyl radicals in a concentration-dependent manner using the fluorescence method, but the visible method could not.Conclusions In contrast to visible method, fluorescence method has the advantages of higher sensitivity and stronger anti-interference ability to the color of test substance and the specificity of solvents.By virtue of large measurement window, it can be applied to evaluating the effect of fat-soluble test substances.