The synthesis of polyhedral oligomeric silsesquioxanes (POSS)-containing conjugated polymer (CP) and the polymer loaded poly(lactic-co-glycolic-acid) (PLGA) nanoparticles (NPs) with surface antibody functionalization for human epidermal growth factor receptor 2 (HER2)-positive cancer cell detection are reported. Due to the steric hindrance of POSS, NPs prepared from POSS-containing CP show improved photoluminescence quantum yield as compared to that for the corresponding linear CP encapsulated NPs. In addition, the amount of -NH2 groups on NP surface is well-controlled by changing the molar ratio of poly(lactic-co-glycolic-acid)-b-poly(ethylene glycol) (PLGA-b-PEG-NH2) to PLGA-OCH3 during NP formulation. Further conjugation of the NH2-functionalized CP NPs with trastuzumab (Herceptin) yields NPs with fine-tuned protein density. These NPs are able to discriminate SKBR-3 breast cancer cells from MCF-7 breast cancer cells and NIH/3T3 fibroblast cells both on substrate and in suspension by taking advantage of the specific binding affinity between trastuzumab and HER2 overexpressed in SKBR-3 breast cancer cell membrane. The high quantum yield and fine-tuned surface specific protein functionalization make the POSS-containing CP loaded NPs a good candidate for targeted biological imaging and detection.