The invention relates to a preparation method of a biological detergent. The biological detergent is prepared by mixing APG (C8-16), maltose, ethanol, composite amino acid, aloe, chitin, salt, probiotics, lactic acid, spice and water. According to the method, the maltose serving as a main raw material, the APG biosurfactant and the probiotics are subjected to biochemical reaction to form various biological resolvase under the auxiliary edge action of the chitin. The process comprises the following steps of: preparing the maltose, the APG and the water according to the percentage; adding the probiotics and the chitin and mixing; stirring uniformly; performing sealed fermentation for 48 to 72 hours; adding the ethanol, the composite amino acid, the aloe, the lactic acid and the spice, and mixing and stirring uniformly; adding the salt and mixing; and standing for 24 hours. The biological detergent has a good pollutant-removing effect and a wide using range, does not damage a human body, protects objects, prolongs the service life of the objects, and is safe and environment-friendly.