Neurofilament light chain (NfL) is released into the peripheral circulation by damaged axons. To evaluate the diagnostic value of serum NfL concentration in dogs with intracranial diseases. Study included 37 healthy dogs, 31 dogs with idiopathic epilepsy (IE), 45 dogs with meningoencephalitis of unknown etiology (MUE), 20 dogs with hydrocephalus, and 19 dogs with brain tumors. Cohort study. Serum NfL concentrations were measured in all dogs using single-molecule array technology. Serum NfL concentration in dogs with each structural disease was significantly higher than in healthy dogs and dogs with IE (P = .01). The area under the receiver operating characteristic curve of NfL for differentiating between dogs with structural diseases and IE was 0.868. An optimal cutoff value of the NfL 27.10 pg/mL had a sensitivity of 86.67% and a specificity of 74.19% to differentiate the dogs with IE from those with structural brain diseases. There were significant correlations between NfL concentrations and lesion size: (1) MUE, P = .01, r = 0.429; (2) hydrocephalus, P = .01, r = 0.563. Serum NfL could be a useful biomarker for distinguishing IE from structural diseases in dogs and predicting the lesion sizes of MUE and hydrocephalus.