L-type amino acid transporter 1 (LAT1)-specific ligands and polyion complexes are used as brain-specific targets to deliver RNA-based drugs across the blood-brain barrier. We characterized a LAT1-targeting antisense oligonucleotide (ASO) encapsulated nanoparticle, “Phe-NPs/ASO.” A 25% density of phenylalanine effectively binds to the surface of LAT1 targeting nanoparticles in the GL261-Luc cells and Phe-NPs/ASO showed higher binding affinity compared to that without phenylalanine by cellular binding assay. To further characterize the blood-brain barrier-targeting effect and tissue distribution following a single-dose intravenous injection in mice, we performed in vivo biodistribution studies using fluorescence imaging. The Phe-NPs/ASO were detected in the brain tissue 1 h post-intravenous at an approximately 64-fold higher ratio than that of the same ASOs administered in the absence of any nanoparticle carrier. The brain tissue delivery of antisense oligonucleotide-loaded Phe-NPs was also confirmed in a fluorescence imaging study performed in non-human primates. These results demonstrate that Phe-NPs may successfully deliver an ASO to the brain tissue across brain regions. Phe-nanoparticles loaded with RNA-based drugs have the potential to treat diseases of the central nervous system, including all forms of neurodegenerative diseases.