Tyrosine kinase receptor B (TrkB) mediates neurotrophic effects of brain-derived neurotrophic factor (BDNF) to increase neuronal survival, differentiation, synaptic plasticity, and neurogenesis. The therapeutic potential of TrkB activation using BDNF has been demonstrated well in several preclinical models of CNS diseases, validating TrkB as a promising drug target. Therefore, we aimed to develop TrkB-specific receptor agonists by using a monoclonal antibody approach. After generation of hybridoma clones and assessment of their binding and functional activity, we identified five mouse monoclonal antibodies that show highly selective binding to TrkB and that induce robust activation of TrkB signaling. Epitope mapping studies using competition analysis showed that each of the monoclonal antibodies recognizes a unique binding site on TrkB, some of which are distinct from BDNF docking sites. These antibodies behave as true agonists based on their ability to both activate proximal and secondary signaling molecules downstream of TrkB receptors and promote neuronal survival and neurite outgrowth. The binding affinities and the functional efficacy of these antibodies are comparable to those of BDNF, whereas they do not bind to the p75 low-affinity neurotrophin receptor at all. Therefore, they could represent novel reagents to explore the pathophysiological roles of TrkB and its potential therapeutic utility in treating CNS disorders.