Accurate ante-mortem diagnosis of non-infectious meningoencephalomyelitis (NIME) in dogs is challenging due to the similarity of clinical presentations, imaging findings, and cerebrospinal fluid (CSF) analysis results with other diseases. This study aimed to apply state-of-the-art quantitative proteomic technology to identify novel biomarkers for NIME. Serum and CSF samples from 11 dogs were included, with the control group consisting of patients presenting with intervertebral disc disease (IVDD, n = 6) and the study group consisting of dogs suffering from NIME (n = 5). Mass spectrometry-based quantitative proteomics revealed a set of 36 proteins with significant differential abundance in CSF samples. Up-regulated proteins in NIME CSF included immunoglobulins, inter-alpha-trypsin inhibitor heavy chain 2, acid sphingomyelinase-like phosphodiesterase, and chitinase 3-like protein 1, all associated with immune response and inflammation. Conversely, significantly down-regulated proteins included neural cell adhesion molecule, contactin-1, and procollagen C-endopeptidase enhancer, which are involved in neurodevelopment and synaptic plasticity. No differences in serum profiles were observed among the groups. This study identified a panel of CSF protein biomarker candidates for NIME and provided new insights into the pathogenesis of the disease, suggesting that neuronal dysfunction and immune dysregulation may be involved.