The invention belongs to the technical field of function materials, and specifically relates to a Raman strengthening active microsphere and a preparation method and an application thereof. The Raman strengthening active microsphere provided by the invention has a core-shell structure, the core of the Raman strengthening active microsphere is a melamine resin microsphere, and the shell is uniform and compact silver nanometer granules. The preparation method comprises the steps of firstly preparing uniform melamine-oxymethylene microsphere through fasculation, then utilizing a reducing agent to restore silver nitrate through a normal position chemical reduction method, and uniformly and compactly depositing the generated silver nanometer granules onto the surface of the melamine resin microsphere, so as to prepare the MF/Ag-NPs composite microsphere. The raman strengthening active microsphere utilizes a single microsphere as a raman strengthening base material, and is used for detecting Raman. The single Raman strengthening active base material microsphere provided by the invention has the characteristics of high stability and high Raman strengthening property. The method provided by the invention has the advantages that the operation is simple, the process is controllable, and the prepared composite microsphere can be used for analyzing and detecting organic contaminants such as pesticide residue of a single microsphere in a Raman manner.