Based on the multivalent binding capability of streptavidin (SA) to biotin, a multifunctional quantum dot probe (QD‐(AS‐ODN+p160)) coupled with antisense oligonucleotide (AS‐ODN) and peptide p160 is designed for real‐time tracking of targeted delivery of AS‐ODN and regulation of folate receptor‐α (hFR‐α) in MCF‐7 breast cancer cells. Fluorescence spectra, capillary electrophoresis (CE) and dynamic light scattering (DLS) are used to characterize the conjugation of AS‐ODN and p160 with quantum dots (QDs), DLS results confirm the well stability of the probe in aqueous media. Confocal imaging and quantitative flow cytometry show that QD‐(AS‐ODN+p160) is able to specifically target human breast cancer MCF‐7 cells. Low temperature and ATP depletion treatments reveal the cellular uptake of QD‐(AS‐ODN+p160) is energy‐dependent, and the effects of inhibition agents and co‐localization imaging further confirm the endocytic pathway is mainly receptor‐mediated. Transmission electron microscopy (TEM) shows the intracellular delivery and endosomal escape of QD probe along with incubation time extended. Two transfection concentrations of QD probe (10 nM and 50 nM) below half inhibitory concentration (IC50) value are chosen according to MTT assay. Real‐time PCR shows at these two concentration cases the relative mRNA expression levels of hFR‐α reduce to 72.5 ± 3.9% and 17.6 ± 1.0%, respectively. However, western blot and quantitative ELISA analysis show the expression level of hFR‐α protein has a significant decrease only at 50 nM, indicating that gene silence is concentration‐dependent. These results demonstrate that the QD‐(AS‐ODN+p160) probe not only achieves gene silence in a cell‐specific manner but also achieves real‐time tracking during AS‐ODN intracellular delivery. A new multifunctional quantum dot (QD) probe is prepared by coupled with anti‐hFR‐α antisense oligonucleotide and targeted peptide p160 with QDs through biotin‐streptavidin interaction. Down‐regulation of hFR‐α in a cell‐specific manner is achieved. The probe is effectively transported into the cytoplasm through receptor‐mediated endocytosis, and finally antisense oligonucleotides combine with hFR‐α mRNA and block the protein translation.