The invention relates to a preparation method of a surface-functionalized molecularly imprinted polymer microsphere. The crosslinking degree of the molecularly imprinted polymer microsphere is above 60 percent, and therefore, the molecularly imprinted polymer microsphere has excellent molecule recognition to a template molecule and the particle size of 1-5 microns and is provided with an active function group on the surface. The molecularly imprinted polymer microsphere is prepared by adopting a new method of controllable/active free radical deposition and polymerization. The invention has the advantages of simple method, wide application range, pure product and the like. The prepared molecularly imprinted polymer microsphere has a wide application potential in mass fields of chromatographic stationary phase, biological sample analysis, medial clinic immunity analysis, food and environment monitoring, analogy enzyme catalysis, biomimetic sensors and the like.