A fluorescence polarization (FP) assay for polysorbate 20 (Tween 20) content in fusion protein injection was developed. The Tween 20 content in samples was determined based on the principle that the intensity of fluorescence polarization at an excitation wavelength of 485 nm, produced by binding of hydrophobic fluorescent dye 5-dodecanoylaminofluorescein (DAF) to the nonpolar core in Tween 20 micelle, was pos. related to the micelle concentration in solution The developed method was verified for specificity, accuracy, precision and goodness-of-fit. The developed method showed high specificity, by which the interference of protein in detection system was eliminated. The samples at high (250 μg/mL), moderate (150 μg/mL), low (50 μg/mL) and LLQQ (lower limit of quantitation, 25 μg/mL) concentrations were determined by the developed method for 3 times, of which the mean recovery rates were within the acceptable range of (100±20) %, both the coefficients of variation in intra-and inter-assays were less than 15%. The standard curve showed a high goodness-of-fit. The developed FP method showed high specificity, accuracy and precision, which might be used for determination of Tween 20 content in protein products and was suitable for monitoring the effective concentration of Tween 20 during investigation on stability.