The invention belongs to the technical field of preparation of animal bioreactors, and particularly relates to a preparation method of a fish skin mucous gland bioreactor and an application of the preparation method. The preparation method comprises the steps of identifying specific expression genes, promoters and protein secreting signal peptide of fish skin mucous gland cells, constructing transgene expression vectors which can specifically express endogenous or heterogenesis bioactive substances in fish skin and mucous gland cells, and developing transgenic fish which can stably inherit andsecrete bioactive substances to fish mucosa, wherein the bioactive substances secreted by mucous glands can be used for growth, stress resistance and disease resistance of animals and plants, healthcare and disease prevention and treatment of human, and enzymes for business. The developed fish skin mucous gland bioreactor disclosed by the invention has the characteristics that breeding and groupextending are easy, much skin mucus secretes, mucus collection is convenient, and the bioactive substances are easy to purify, and large-scale production and high-efficiency application of the fish skin mucous gland bioreactor can be realized.