Detection of α-synuclein (α-syn) amyloid seeds in human biofluids has attracted great interest for clinical diagnosis of synucleinopathies. However, as a common biomarker, α-syn lacks specificity in reliably differentiating distinct disorders. Here, we report tubulin polymerization promoting protein (TPPP/p25) as a cerebrospinal fluid (CSF) biomarker for the specific diagnosis of multiple system atrophy (MSA). We demonstrate that native TPPP/p25 is self-protected against amyloid aggregation, while disease-related mutation disrupts this protection, triggering TPPP/p25 aggregation. Cryo-electron microscopy (cryo-EM) analysis reveals that the well-folded core domain (CORE) undergoes large conformational changes to mediate amyloid formation. Based on this insight, we developed a seed amplification assay using a minimized CORE (miniCORE) monomer, which detects TPPP/p25 amyloid seeds in CSF and robustly differentiates MSA from Parkinson's disease (PD) and other neurodegenerative diseases. Our findings establish misfolded TPPP/p25 as a promising, specific biomarker in biofluids for MSA diagnosis.