Nano-selenium synthesized by the probiotic bacterium Lactobacillus plantarum LP21 was analyzed by transmission electron microscopy (TEM) and energy spectroscopy, and its particle size was measured by a particle size analyzer. Additionally, biomolecule distribution on the surface of nano-selenium were analyzed by Fourier transform infrared (FTIR) spectroscopy, and the antibacterial activity of nano-selenium against Vibrio alginolyticus was tested. The results showed that the nano-selenium was spherical in shape and well dispersed; the average particle size of the purified nano-selenium was 184.6 nm, and its surface was covered with proteins, polysaccharides and other biological molecules, showing an amorphous form. The antibacterial activity of nano-selenium against V. alginolyticus and Escherichia coli was strong, and the minimum inhibitory concentration (MIC) against V. alginolyticus was 286 μg/mL. The growth, motility and biofilm of V. alginolyticus were significantly inhibited by the nano-selenium at sub-inhibitory doses, and the cell morphology was changed as well. The results of this study provide a basis for its application to control pathogenic infection in aquaculture.