epsilon-Poly-L-lysine (epsilon-PL) consists of 25-35 lysine residues which are linked by an isopeptide bond formed by dehydration condensation of alpha-carboxyl and epsilon-amino groups and has good antibacterial activity and broad-spectrum inhibition range. However, there is no clear conclusion about the structure and antibacterial mechanism of epsilon-PL in aqueous solution. Herein, a high purity of epsilon-PL was prepared using Amberlite IRC-50 ion-exchange resin. Membrane filtration and dynamic light scattering were used to study the variations of epsilon-PL aggregation in aqueous solution with pH value. The conformational changes and antibacterial activities of epsilon-PL and carbamoylated epsilon-PL in different water environments were studied with circular dichroism (CD) and inhibition zone. The structural changes during the spray-drying process were determined by Fourier transform infrared spectroscopy. The results indicated that the side chain amino charge played a decisive role in the epsilon-PL conformation and aggregation. epsilon-PL exhibited the properties of a beta-sheet during spray drying from acidic liquids to solids. The cation enhanced the antibacterial activity of epsilon-PL but did not play a key role. Instead, the backbone of epsilon-PL might determine the mechanism of epsilon-PL antibacterial.