Ubiquitin, a 76-residue polypeptide, functions as a post-translational modifier and forms polyubiquitin (polyUb) chains on substrate proteins to activate diverse cellular functions. Ubiquitin is often found in the lesions associated with neurodegenerative disorders like Alzheimer's and Huntington's disease. It was recently observed that polyubiquitin chains, under in vitro conditions, often form β-sheet-rich fibrillar amyloid aggregates. However, the exact mechanism of conversion from monomer polyUb to amyloid-like fibrils is poorly understood. We report that polyubiquitin forms higher-order oligomeric fibrils and temperature, pH, and ionic strength modulate polyubiquitin (polyUb) fibrillation. Cosolutes such as ionic liquids and macromolecular crowding agents also enhance the aggregation rate. These findings uncover that temperature/cosolutes lower the energy barrier to populate a partially unfolded form (M*), which drives amyloid-like fibril formation. Other factors increase the effective protein concentration of these partially unfolded forms, accelerating aggregation. These insights provide the fundamental basis for ubiquitin chain aggregation, which is necessary to guide the development of therapies and diagnostics.