Neurotrophins (NTs) play essential roles in the development and survival of neurons in PNS and CNS. In the cochlea, NTs [e.g., NT-3, brain-derived neurotrophic factor (BDNF)] are required for the survival of spiral ganglion neurons (SGNs). Preservation of SGNs in the cochlea of patients suffering sensorineural deafness caused by loss of hair cells is needed for the optimal performance of the cochlear implant. Directly applying exogenous BDNF into the cochlea prevents secondary degeneration of SGNs when hair cells are lost. However, a common translational barrier for in vivo applications of BDNF is the poor pharmacokinetics, which severely limits the efficacy. Here we report that 7,8-dihydroxyflavone and 7,8,3'-trihydroxyflavone, both small-molecule agonists of tyrosine receptor kinase B (TrkB), promoted SGN survival with high potency both in vitro and in vivo. These compounds increased the phosphorylated TrkB and downstream MAPK and protected the SGNs in a TrkB-dependent manner. Their applications in the bulla of conditional connexin26 null mice offered significant protection for SGN survival. The function of survived SGNs was assessed by measuring evoked action potentials (APs) in vitro and electrically evoked auditory brainstem response (eABR) thresholds in vivo. APs were reliably evoked in cultured single SGNs treated with the compounds. In addition, eABR thresholds measured from the treated cochleae were significantly lower than untreated controls. Our findings suggest that these novel small-molecule TrkB agonists are promising in vivo therapeutic agents for preventing degeneration of SGNs.