$varepsilon$-Poly-L-lysine ($varepsilon$-PLL) produced by Streptomyces albulus CGMCC 1986 was fractionated using ultra-filtration technique with 2 and 5 kDa cut-offs of membrane. The number-average molecular weight of each fraction was determined by $^1H$ nuclear magnetic resonance (NMR) method. The number-average molecular weights of the cutoffs of 5 and 2 kDa and the filtrate are 4,230.95, 3,687.80, and 1,900.82 Da, respectively. $^1H$ NMR indicates the chemical shifts of $alpha$-H, $eta$-H, $gamma$-H, $delta$-H, and $varepsilon$-H are very similar to all the fractions. Fourier transform-infrared (FTIR) spectra showed that the $varepsilon$-PLL solid samples obtained by freeze-drying at pH 5 with molecular weights higher than 2 kDa take on a $eta$-turn conformation, however, the fraction with molecular weight smaller than 2 kDa adopts random coil structure. The antibacterial test proved that the fraction between 2 and 5 kDa of membranes behaves the highest antibacterial activity than other fractions for the test strains of Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, and Shigella.