The objective of this research was to construct the gene-targeted polymeric microvesicles (PMVs) and investigate their In Vitro ability to bind specifically to human epidermal growth factor receptor 2 (HER-2) (+) breast cancer (BC) cells. PMVs were formed using a block copolymer, methoxy polyethylene glycol-poly(L-lactide) (mPEG-PLLA), as the shell and encapsulating liquid perfluoropentane. Plasmid DNA and biotinylated HER-2 monoclonal antibody were conjugated to form the gene-loaded HER-2-targeted PMVs for BC cells. The characterization, physicochemical properties, and antibody coupling efficiency of the PMVs were evaluated. The PMVs were then co-cultured with HER-2 (+) BT474 cells, and their ability to target and bind to HER-2 (+) BC cells was observed under a microscope. Results revealed that the average particle size (APS) of the gene-targeted PMVs was (3.92±1.01) μm, with a uniform particle size distribution (PSD), smooth and transparent surfaces, and superior stability. The fluorescence intensity (FI) of PMVs in Group A was higher (16 vs. 9) to that in Group B, indicating a high binding rate (BR) (97.01%) between the PMVs and HER-2 monoclonal antibody. BT474 cells exhibited green fluorescence on their surface, which was stronger than that observed in SK-BR-3 cells, while no obvious green fluorescence was visualized in MDA-MB-231 cells or Hs578Bst cells. PMVs in Group A presented extensive binding to BT474 cells, mainly distributed on the cell membrane and surrounding areas. Only a few PMVs in Groups B and C were observed to bind to BT474 cells. In conclusion, the gene-loaded HER-2-targeted PMVs exhibited excellent stability and high specificity for binding to HER-2 (+) BC cells In Vitro, suggesting their potential application value.