The invention discloses dopamine and dopamine metabolite based molecularly imprinted magnetic nanospheres as well as a preparation method and an application thereof. The method comprises the followingsteps: synthesizing carboxyl-functionalized magnetic nanospheres with a hydrothermal method; adding the carboxyl-functionalized magnetic nanospheres and two epinephrine metabolites to a phosphate buffer solution in a certain ratio to obtain a dual-template-carrier complex; stirring a separation product, functional monomer hyperbranched polyethyleneimine and crosslinking agent glutaraldehyde in the phosphate buffer solution for polymerization to obtain a polymer; separating a solid polymer out by externally applying a magnetic field; finally, performing reflux elution on the solid polymer, andperforming vacuum drying to obtain the dopamine and dopamine metabolite based molecularly imprinted magnetic nanospheres. The preparation method is simple, conditions are mild, and the prepared molecularly imprinted magnetic nanospheres are uniform in particle size, high in stability and good in water solubility, and can realize selective enrichment and detection of dopamine and 3,4-dihydroxyphenylacetic acid in complicated matrix of human fluid simultaneously and rapidly.