This study established an approach to rapidly determine the Tween-80 in traditional Chinese medicine (TCM) injection by using near-infrared (NIR) spectroscopy. Totally 133 standard solutions of Tween-80 were prepared and randomly divided into calibration set and validation set, containing 109 and 24 samples, respectively. Spectral data were preprocessed and then subjected to establish a predictive model using partial least-squares (PLS). The standard error of cross validation (SECV), standard deviation of calibration (SEC), and the determination coefficient (R) of the established model were 0.0561, 0.0526, and 0.9986, respectively. The model was successfully applied to determine Tween-80 contents in 25 XBJ Injections and 40 FFSX Injections, and it produced satisfactory quantitative analysis results with average relative deviations 0.60% and 0.16%, respectively, for 25 XBJ Injections and 40 FFSX Injections, and the maximum relative deviations 8.57% and 7.60%, respectively. This work shows that NIR model displayed quite good predictive ability for Tween-80 quantitative analysis, which could potentially be applied to rapid determination of Tween-80 in the production process of TCM injections and other TCM products.