The invention discloses preparation of a biological film cleaner and an application of the cleaner. The biological film cleaner is prepared from, in weight percentage, 45-55% of quorum sensing inhibitors, 25-35% of surface active agents and 15-20% of water softeners. The quorum sensing inhibitors are prepared by mixing, in weight percentage, 30-40% of pineapple-ketone, 30-40% of menthol and 30-40% of vanillic aldehyde. The quorum sensing inhibitors serve as main components, biological film generation of pathogenic bacteria and growth of bacteria are inhibited by inhibiting quorum sensing behaviors of microorganisms instead of resisting and killing the bacteria by compounds according to existing most cleaners, and the problem of drug resistance in the bacteria killing process is avoided.