Neurotrophic factor 3 (NTF3) is a cysteine knot protein and a member of the nerve growth factor (NGF) family of cytokines. NTF3 engages the Trk family of receptor tyrosine kinases, playing a pivotal role in the development and function of both the central and peripheral nervous systems. Its involvement in neuronal survival, differentiation, and growth links NTF3 to a spectrum of neurodegenerative diseases. Consequently, targeting NTF3 with antibodies holds promise as a first in class therapeutic opportunity for a wide range of conditions. Specific and neutralizing antibodies against NTF3 were successfully isolated using phage display. Initial phage display selections revealed a preference of hits for a longer than average complementarity-determining region 3 (CDR3) in the heavy chain variable domain (VH). To investigate this further we developed a long loop length VH CDR3 antibody library that demonstrated increased hit rates versus a standard antibody library and allowed the isolation of IgG that demonstrated inhibition of functional activity, coupled with a favourable kinetic profile. Structural analysis of the Fab/NTF3 interaction, via X-ray crystallography, unveiled an unconventional interaction wherein regions beyond the longer CDR loops of the Fab induced ordering in a flexible loop on NTF3, which remained disordered in its free antigenic state. This comprehensive approach not only sheds light on the therapeutic potential of NTF3-specific antibodies but also provides critical structural details that enhance our understanding of the complex NTF3-Fab interaction thus offering valuable insights for future antibody design and therapeutic development.