Congenital hearing loss is a common disorder and over 60 mutations in the sensory hair cell protein, otoferlin, have been linked to pre-lingual deafness. Although otoferlin is essential for hearing, the large size and low solubility of the protein have limited approaches to study its function on a molecular-level. To overcome these challenges, we have developed single-molecule fluorescence assays which have allowed us to quantitatively probe otoferlin function. Based upon our findings, we have shown that otoferlin acts as a calcium-sensitive scaffold for other membrane-fusion proteins at the plasma membrane. The ability to localize fusion proteins at the site of calcium-influx would reduce synaptic delay and allow for rapid neurotransmitter release in response to a sound stimulus. Second, we have directly shown that otoferlin inserts into lipid-bilayers. This finding is significant because insertion into the membrane bilayer has been demonstrated to be a crucial step for mediating the fusion of synaptic vesicles to the plasma membrane.