Objective The aim of this study was to explore the ototoxicity of toluene in the early development of zebrafish embryos/larvae. Methods Zebrafish were utilized to explore the ototoxicity of toluene. Locomotion analysis, immunofluorescence, and qPCR were used to understand the phenotypes and molecular mechanisms of toluene ototoxicity. Results The results demonstrated that at 2 mmol/L, toluene induced zebrafish larvae death at 120 hours post fertilization (hpf) at a rate of 25.79％ and inhibited the rate of hatching at 72 hpf. Furthermore, toluene exposure inhibited the distance travelled and average swimming velocity of zebrafish larvae while increasing the frequency of movements. As shown by fluorescence staining of hair cells, toluene inhibited the formation of lateral line neuromasts and middle line 1 (Ml1) neuromasts in 3 days post fertilization larvae in a concentration-dependent manner. Toluene altered the expression level of genes involved in ear development/function in zebrafish, among which the mRNA levels of cd164l2, tekt3, and pcsk5a were upregulated, while the level of otofb was downregulated, according to the qPCR results. Conclusion This study indicated that toluene may affect the development of both the inner ear and lateral line systems in zebrafish, while the lateral line system may be more sensitive to toluene than the inner ear.