Background: Currently, no tests can definitively diagnose and distinguish neuromyelitis optica spectrum disorder (NMOSD) from multiple sclerosis (MS). Methods: Initially, cerebrospinal fluid (CSF) proteomics were employed to uncover the novel biomarkers that differentiate NMOSD from MS into cohorts of 10 MS and 10 NMOSD patients. Subsequently, screening biomarkers were validated using an enzyme-linked immunosorbent assay method and CSF and serum samples from 20 MS patients, 20 NMOSD patients, 20 non-inflammatory neurological controls, and 20 healthy controls. Results: In study cohort, insulin-like growth factor-binding protein 7 (IGFBP7) and lysosome-associated membrane glycoprotein 2 (LAMP2) were screened. In validation cohort, serum and CSF IGFBP7 not only exhibited higher levels in MS and NMOSD patients than controls, but also had greatest area under the curve (AUC, above or equal to 0.8) in MS and NMOSD diagnoses. Serum IGFBP7 (0.945) and CSF IGFBP7 (0.890) also had the greatest AUCs for predicting MS progression, while serum LAMP2 had a moderate curve (0.720). Conclusions: IGFBP7 was superior in diagnosing MS and NMOSD, and IGFBP7 and serum LAMP2 performed exceptionally well in predicting the MS progression. These results offered reasons for further investigations into the functions of IGFBP7 and LAMP2 in MS and NMOSD.