The invention provides an immobilized enzyme, a preparation method therefor and application of the immobilized enzyme. The immobilized enzyme comprises activated polyethylene imine (PEI) and an enzymecovalently bound onto the activated PEI; the enzyme is selected from any one of the following enzymes: transaminase, ketoreductase, cyclohexanone monooxygenase, phenylalanine ammonia lyase, ene reductase, imine reductase, amino acid dehydrogenase and nitrilase. The enzyme and the PEI are immobilized, and the enzyme is immobilized between a poly-net structure formed by the PEI, so that the mechanical stability is improved, immobilization of the enzyme is realized, and the situation that the activity of the enzyme is reduced due to direct covalent bonding of the enzyme and cross-linking agentsincluding glutaraldehyde and the like during immobilization is avoided.