Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of central nervous system (CNS) biologics. We established a functional selection method for BBB-penetrating VNAR antibodies from a synthetic phage library. Combining in vitro selection on human TfR1 followed by in vivo selection in mice identified the species cross-reactive VNAR TXB2. As a Fc fusion protein, TXB2 binds brain capillaries after intravenous injection and is rapidly transported into the parenchyma and subsequently accumulates in TfR1-positive neurons throughout the brain. Doses of 1.875 mg/kg (25 nmol/kg) produced a rapid, sustained CNS exposure and robust pharmacological activity when fused to neurotensin. There was no was evidence of target-mediated clearance, TfR1 depletion or cytotoxicity as seen with various antibodies to TfR1. This functional selection method for VNARs that can shuttle molecules across the BBB with high efficiency and species cross-reactivity should be widely applicable to other BBB transport systems.A one-sentence summary Development of highly efficient, TfR1 specific, cross-species reactive blood-brain barrier (BBB) shuttle based on shark single domain VNAR antibody.* (TfR1) : Transferrin Receptor 1, (BBB) : blood-brain barrier, (Tf) : Transferrin, (CNS) : central nervous system, (VNAR) : Variable domain of New Antigen Receptors, (CDR3) : complementarity-determining region 3, (RT) : room temperature, (SEC) : size exclusion chromatography, (HSA) : human serum albumin, (NT) : Neurotensin, (IHC) : Immunohistochemistry, (NGS) : next generation sequencing, (PK) : Pharmacokinetic, (BCSFB) : blood-CSF barrier, (%ID) : Percentage injected dose, (AUC) : Area under the curve, (AEF) : attenuated effector function, (SDS-PAGE) : sodium dodecyl sulfate polyacrylamide gel electrophoresis, (WB) : Western blot, (IV) : intravenous