The invention relates to a method for preparing a nano-cellulose antibacterial composite material through on-line culture. The method comprises the following steps of: (1) inoculating an activated bacterial cellulose producing strain into a liquid culture medium, performing amplification culture, transferring the liquid culture medium into a bioreactor, culturing, adding an antibacterial material into the liquid culture medium, and continuing to culture to obtain an unpurified antibacterial bacterial cellulose composite material; and (2) peeling a bacterial cellulose membrane from a framework or directly soaking the composite material in a NaOH solution, treating in water bath, and washing until an obtained product is neutral to obtain the nano-cellulose antibacterial composite material. The preparation efficiency is high, and the method is simple, convenient, feasible and low in cost; and the surface of the bacterial cellulose composite material has a nanoscale three-dimensional fibrous mesh-shaped structure, the tensile strength of the material is greatly improved compared with that of a pure bacterial cellulose membrane, and the material can be widely applied to products such as facial masks, wound dressings, plasters, artificial skins and the like.