Abstract: The fermentation supernatant of Bacillus velezensis DH82 was precipitated with optimal 70% saturation of ammonium sulfate and the crude protein was obtained by dialysis. The crude protein was separated by Sephadex G-75 column. The chromatographic conditions were set at 1.6 cm×50.0 cm column, loading sample volume 5 mL, using ultrapure water as eluting buffer, flowing rate 2 mL/min and detecting proteinat wavelength of 280 nm. Two peak proteins PrI and PrII were obtained. The result of inhibition spectrum analysis showed that the proteins had good antimicrobial activity against the aquatic pathogens such as Edwardsiella tarda, Aeromonas hydrophila, Vibrio campbellii, V. alginolyticus, V. harveyi, Streptococcus agalactiae and Staphylococcus aureus. The antimicrobial active substance of B. velezensis DH82 had a broad inhibition spectrum for some aquatic pathogenic bacteria. The protein PrII showed higher antimicrobial activity against the plant pathogen fungi of Fusarium equisetum. The minimal inhibitory concentration (MIC) of the peak proteins was determined. The MICs of the proteins to E. tarda and V. alginolyticus were lower than 7.16 μg/mL. The findings in this study indicate that B. velezensis DH82 is valulble in the potential application on the prevention and control of common bacterial diseases in aquaculture and some cases of plant fungal diseases.