Targeting is vital for precise positioning and efficient therapy, and integrated platforms for diagnosis and therapy have attracted more and more attention. Herein, we established dual-template molecularly imprinted polymer (MIP) coated fluorescent silicon nanoparticles (Si NPs) by using the linear peptide of the extracellular region of human epidermal growth factor receptor-2 (HER2) and adopting doxorubicin (DOX) as templates for targeted imaging and targeted therapy. Benefiting from the epitope imprinting approach, the imprinted sites generated by peptides on the MIP surface can be employed for recognizing the corresponding protein, which allowed the MIP to specifically and actively target HER2-positive breast cancer cells. Because of its ability to identify breast cancer cells, the MIP was applied for targeted fluorescence imaging by taking advantage of the excellent fluorescence properties of Si NPs, and the DOX-loaded MIP (MIP@DOX) can act as a therapeutic probe to effectively target and kill breast cancer cells. In fluorescence images, the targeting of the MIP promoted more uptake of the nanoparticles by cells than the non-imprinted polymer (NIP), so HER2-positive breast cancer cells incubated with the MIP exhibited stronger fluorescence, and there was no significant difference in fluorescence when HER2-negative cells and normal cells were respectively hatched with the MIP and NIP. Importantly, the cell viability was evaluated to demonstrate targeted accumulation and therapy of MIP@DOX for breast cancer cells. The nanoplatform for diagnosis and therapy combined the high sensitivity of fluorescence with the high selectivity of the molecular imprinting technique, which holds vital potential in targeted imaging and targeted therapy in vitro.