The invention discloses a preparation method of carbofuran molecularly imprinted microspheres, comprising the following steps of: dissolving template molecules and a functional monomer into a pore-forming agent solution containing a cross-linking agent and an initiator so as to obtain a reaction mixture, carrying out ultrasonic degassing, sparging nitrogen for deoxygenation, carrying out polymerization in a sealed state, carrying out centrifugation after complete reaction so as to obtain a white powdery polymer, carrying out Soxhlet extraction respectively by the adoption of a methanol acetate mixed liquor and methanol as extraction solvents, drying under vacuum, and removing the solvents so as to obtain the carbofuran molecularly imprinted microspheres. The invention also discloses the carbofuran molecularly imprinted microspheres prepared by the preparation method and an application thereof. The carbofuran molecularly imprinted microspheres obtained in the invention has specific selectivity for carbofuran in agricultural products, can be used to detect residual pesticides on agricultural products rapidly, sensitively, accurately and efficiently, and also can be used as a solid-phase extraction filler.