The aim of this work was to produce antimicrobial protein using novel endophyte DNN6. The endophyte DNN6 that showed distinct antibacterial activity was isolated from Laminaria japonica Aresch in Dalian China. The strain DNN6 was identified as Bacillus licheniformis. The orthogonal matrix method was used to optimize the culture conditions for antibacterial activity of strain DNN6, and the optimum conditions for DNN6 were cultivated in 50 g/L sucrose and 10 g/L peptone with pH 9 at 25^°C for 6 d. To obtain the antibacterial protein, different saturation of ammonium sulfate was used. The high yield (2.866 g/L) and antibacterial activity of protein from strain DNN6 was precipitated by 95% saturation of ammonium sulfate. Furthermore, the antifungal activity was detected, and this protein demonstrated some degree of inhibition to all the 10 funguses. The protein of strain DNN6 is potential for application as an antimicrobial substance.