Abstract: The present invention discloses a separation membrane based on a reactive support layer, a preparation method and an application, and belongs to the technical field of water treatment membranes. The preparation method includes: (1) subjecting a porous support membrane to an amino grafting reaction to prepare a reactive support membrane; and (2) subjecting an aqueous phase solution and an oil phase solution containing a multi-membered acyl chloride oil phase monomer to interfacial polymerization on the reactive support membrane to prepare a separation membrane based on a reactive support layer; where an aqueous phase monomer in the aqueous phase solution is a semi-aromatic amine or an aromatic amine. In the present invention, the porous support membrane is subjected to the amino grafting reaction to obtain the reactive support membrane, and the interface polymerization is carried out on a surface of the reactive support membrane.