Abstract: ε-poly-L-lysine (ε-PL) is a natural preservative with good antibacterial effect and high temperature resistance, which has a good application prospect in food. ε-PL is a similar amino acid polymer formed by L-lysine monomer by synthesis-catalytic condensation of α-COOH and ε-NH₂. This polymer was isolated by Japanese scholars Shima and Sakai in the fermentation broth of Small White Streptomyces Streptomyces albulus 346 in 1977. The free amino group on the ε-PL backbone facilitates it to exhibit cationic properties in acidic to weakly alkaline environments. Therefore, ε-PL is able to bind to anions on the cell membrane surface and damages cell membranes of microbes, including Gram-positive and Gram-negative bacteria, fungi and yeast. In 2014, China officially approved its use for the preservative and preservation of starch products, baked foods, meat products, beverages, condiments and other foods. ε-PL has become the third kind of microbial source natural food preservative approved in China. The trypan blue method was established for the determination of ε-PL in the fermentation broth. The side chain of ε-PL is reported to contain large amounts of amino cation, while trypan blue contains a sulfonate anion. According to the precipitation principle of ε-PL and methyl orange, it was found that ε-PL and trypan blue were also able to form a complex and precipitate in the range of pH 4-10. After centrifugation, the supernatant showed a good linear relationship between its absorbance value and the ε-PL concentration at 600 nm. Trypan blue reagent was used as the chromatic reagent. Several factors including the concentration of chromatic reagent, reaction temperature, time, and pH, were investigated to optimize the test conditions. The optimal reaction conditions were determined as follows:the concentration of trypan blue was 3 g/L, pH 6.0, reaction temperature was 50℃ and reaction time was 40 min, the standard curve was Y=-0.012 8X+3.148 8. The calibration curve was linear in the concentration range of 10-100 mg/L. The linear correlation coefficient (R²) of ε-PL working curve was 0.999 8. The LOD and LOQ were 2.03 mg/L and 6.14 mg/L. The coefficient of variation (CV) of the measurement results of the standards with different concentrations was less than 2%. The determination results were not affected by the substrate in the fermentation broth, and the detection time could be effectively shortened. The establishment of this method is of great significance to breeding high yield of ε-PL producing strain.