Intracellular bacterial infections are extremely difficult to be treated because intracellular bacteria have developed resistant mechanisms to escape the immune attack and antibiotic therapy. It remains challenging to develop antibiotic-free materials and relative strategies for treating intracellular bacterial infections. Herein, a new host-defense peptide-mimicking polymer nanoparticle, inspired by cell-penetrating peptides, was developed to eradicate intracellular bacteria by its outstanding antibacterial and pro-inflammatory immunomodulatory. The polymer nanoparticle (TPE-Parg) was prepared through ring-opening polymerization of N6-carbobenzoxy-l-lysine N-carboxyanhydride (Cbz-l-Lys NCA) using 1-(4-aminophenyl)-1,2,2-triphenylethene (TPE-NH2) as the initiator, followed by deprotection of the Cbz-l-Lys NCA group and guanidinium modification. The impact of cationic functional groups and chain length variation on the antibacterial activity of polymer nanoparticle were investigated in detail. The results confirmed that the optimal polymer nanoparticle could not only image bacteria with aggregation-induced blue fluorescence, but also kill planktonic bacteria with low cytotoxicity. Furthermore, the nanoparticle could induce macrophages to generate nitric oxide (NO) and activate the immune system to eliminate intracellular bacteria. The nanoparticle further showed its potent in vivo antibacterial activity in an intracellular Staphylococcus aureus infection model fabricated on mice hypodermic. The obtained multifunctional host-defense peptide-mimicking polymer nanoparticles with potent antibacterial activity (chemotherapy) and pro-inflammatory immunomodulatory (immunotherapy) are excellent alternatives for intracellular antibacterial therapy and provide a direction for developing innovative antimicrobials.