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Abstract The heterogeneous nature of cancers renders the efficacy of the therapeutic treatment diversified. To address this issue, a theranostic approach was proposed by development of a hybrid peptide for simultaneous diagnosis and therapy of cancer cells. Peptide A was composed of the biotinylation site (BS), the HER2/neu-binding motif, and the dockerin domain (Doc). Peptide B was designed with BS and the cohesin domain (Coh). Individually expressed in Escherichia coli, peptide A and peptide B were isolated and then functionalized by conjugation with quantum dots (QDs)-streptavidin and magnetic nanoparticles (MNPs)-streptavidin, respectively. A hybrid peptide was assembled by coupling of the QDs-peptide with the MNPs-conjugated peptide via the Doc-Coh interaction. By administration of the hybrid peptide, HER2/neu-positive breast cancer cells were detected and destroyed as a result of the QDs-emitted fluorescence and the MNPs-mediated hyperthermia.