Development of effective ways to specifically and reversibly block the activity of an enzyme is highly desirable for enhancing the selectivity of enzyme assays. Here we demonstrate a novel approach for selective detection of enzyme activities in complex biological samples by using tailor-made nanoparticles. Employing deoxyribonuclease I (DNase I) as a model enzyme template, we prepared surface imprinted polymers over magnetic nanoparticles with monomers screened out of commonly used functional monomers. The resultant Fe3O4@MIP nanoparticles can not only block the activity of the target enzyme via selective adsorption but also quantitatively release the bound enzyme under mild conditions with the assistance of metal ion cofactors, which offers a very useful tool for enhancing the selectivity in enzyme detection. The approach enables sequential detection of the activities of 3'-5' exonuclease and DNase I in cell lysates. The strategy may be further extended to the detection of other enzyme proteins.