Systemic delivery of therapeutics into the brain is greatly impaired by multiple biological barriers─the blood-brain barrier (BBB) and the extracellular matrix (ECM) of the extracellular space. To address this problem, we developed a combinatorial approach to identify peptides that can shuttle and transport across both barriers. A cysteine-constrained heptapeptide M13 phage display library was iteratively panned against an established BBB model for three rounds to select for peptides that can transport across the barrier. Using next-generation DNA sequencing and in silico analysis, we identified peptides that were selectively enriched from successive rounds of panning for functional validation in vitro and in vivo. Select peptide-presenting phages exhibited efficient shuttling across the in vitro BBB model. Two clones, Pep-3 and Pep-9, exhibited higher specificity and efficiency of transcytosis than controls. We confirmed that peptides Pep-3 and Pep-9 demonstrated better diffusive transport through the extracellular matrix than gold standard nona-arginine and clinically trialed angiopep-2 peptides. In in vivo studies, we demonstrated that systemically administered Pep-3 and Pep-9 peptide-presenting phages penetrate the BBB and distribute into the brain parenchyma. In addition, free peptides Pep-3 and Pep-9 achieved higher accumulation in the brain than free angiopep-2 and may exhibit brain targeting. In summary, these in vitro and in vivo studies highlight that combinatorial phage display with a designed selection strategy can identify peptides as promising carriers, which are able to overcome the multiple biological barriers of the brain and shuttle different-sized molecules from small fluorophores to large macromolecules for improved delivery into the brain.