Purpose Peptide-based therapy is a promising strategy for cancer treatment because of its low drug resistance. However, the major challenge is their inability to target cancer cells specifically. So, a targeted nano-delivery system that could deliver therapeutic peptides selectively to cancer cells to stimulate their action is highly desirable. This study aims to deliver the antitumor peptide, Pep5, to breast tumor cells selectively using a targeting peptide functionalised multi-layered PLGA-PEI nanoparticles. Methods In this study, Pep5 entrapped PLGA-PEI (Pep5-PPN) dual layered nanoparticles were developed. These nanoparticles were decorated with TKD (Pep5-TPPN) on their surface for site-specific delivery of Pep5 to breast tumor cells. The particles were then characterized using various instrumental analyses. In vitro cytotoxicity of the particles was evaluated in estrogen receptor positive (ER +ve ) and triple negative breast cancer (TNBC) cells. An ex vivo tumor spheroid model was used to analyze the antitumor activity of the particles. Results Uniformly round Pep5-TPPN particles were synthesized with an average diameter of 420.8 ± 14.72 nm. The conjugation of PEI over Pep5-PLGA nanoparticles shifted the zeta potential from −11.6 ± 2.16 mV to +20.01 ± 2.97 mV. In vitro cytotoxicity analysis proved that TKD conjugation to nanoparticles enhanced the antitumor activity of Pep5 in tested breast cancer cells. Pep5-TPPN induced cytoskeletal damage and apoptosis in the tested cells, which showed that the mechanism of action of Pep5 is conserved but potentiated. Active targeting of Pep5 suppressed the tumor growth in ex vivo spheroid models. Conclusion A multi-layered nanoparticle functionalized with dual peptide was fabricated for active tumor targeting, which stimulated Pep5 activity to reduce the tumor growth in vitro and ex vivo.