Polysorbate 80, also known as Tween 80, is an amphipathic non-ionic surfactant commonly used as excipient in the food, personal care and pharmaceutical industries. Particularly, it has been widely used in Traditional Chinese Medicine (TCM) injections to enhance solubility and clarity. In recent years, more attentions have benn paid to the quality and application of polysorbate 80 due to incidence of adverse reactions. Some declared that adding of polysorbate 80 might increase the side effects. To avoid abuse of this excipient, it is essential to control the levels of polysorbate 80 in therapeutic formulations within a target level. Assay of polysorbate 80 in TCM injections has become a hot and difficult issue nowadays. Content of polysorbate 80 in preparations could be quantified directly by spectrophotometry, size-exclusion chromatography coupled with evaporative light scattering detector (SEC-ELSD) or liquid chromatography combined with mass spectrometry (LC-MS) method. Acid hydrolysis followed by high performance liquid chromatography coupled with ultraviolet detector (HPLC-UV) or gas chromatography (GC) determination was also used as an indirect measurement of polysorbate 80. But it's hard to find a unified conversion formula or reference standard for accurate quantification owing to the fact that the surfactant is a complex mixtures of oligomers, whose composition and ratio of chemical components vary by different manufacturers. In addition, it becomes a challenge to measure polysorbate 80 in TCM injections without false-positive interference due to the complex matrix of the preparation. Taking Shengmai Injection as example, a novel method for determination of polysorbate 80 in TCM injections was proposed based on absorption coefficient, which offered a solution for the above problems. After optimization of detection wavelength, chromogenic reagent and of the shaking and standing time of the liquid-liquid extraction, absorption coefficient (E1%1 cm) of cobalt thiocyanate complex of polysorbate 80 was obtained from 6 different bands of instruments as 104.23 with RSD of 2.08%. Shengmai Injection was first diluted by 10 times. To 1.0 mL of the test solution, 10 and 20 mL of dichloromethane were added accurately to form a polysorbate/thiocyanate complex, followed by cyclotron oscillation for 3 minutes. The mixture was transferred into a separating funnel and was stranded for 30 minutes. The first 1 mL of the lower layer was discarded and the next 15 mL was collected. Then the absorbance was measured at 320 nm. Finally the content of polysorbate 80 was calculated by using the previously obtained absorption coefficient based on Lambert-Beer law. With no interference from the negative sample, the established method provided precision and reproducibility (% relative standard deviation) less than 3% and accuracy (% spike recovery) of 98.42%. To further validate the accuracy of the method, 10 batches of Shengmai Injection from 2 manufacturers were determined by both the absorption coefficient method and the standard curve method, and the results were compared with the actual feeding content of polysorbate 80 in the sample. Results of paired t test indicated that there were no significance difference between the results obtained by two methods, or between the results of the absorption coefficient method and the actual feeding content of polysorbate 80 (p>0.05). The investigation selected 320 nm as the detection wavelength to improve the sensitivity, which was never adopted in previous work. In this way, remarkable decrease of matrix interference was achieved, thus the problem of misfit determination results and the actual feeding content of polysorbate 80 was solved. With no need for reference standard or standard curve, the search offered a feasible tool for test standard of polysorbate 80 in TCM injection. All in all, the proposed method was sensitive, accurate, rapid and simple, which can provide key constants and new ideas for quality control of preparations containing polysorbate 80.