Abstract: Neurofilament proteins (NFs), defined as a type Ⅳ intermediate filaments, are important components of the neuronal cytoskeleton. They play a key physiological role in maintaining the structural integrity and plasticity of axons and in ensuring the axonal transport function. Under pathological conditions, NFs detach from axons and undergo abnormal aggregation, causing axonal transport dysfunction. In addition, some components of the detached NFs leak into the peripheral circulation system. In patients with amyotrophic lateral sclerosis (ALS), the concentration of NFs is significantly elevated in the cerebrospinal fluid and blood, and the changes in NFs concentration is significantly positively correlated with the disease progression of ALS, suggesting the potential of NFs being used as early diagnostic biomarkers for ALS. In this review, we explored the relationship between NFs structure, assembly, and physiological function, focusing on the molecular mechanisms and clinical manifestations of ALS caused by abnormal assembly of NFs. We comprehensively summarized recent advances in the application of NFs as a new humoral biomarkers for early diagnosis and therapeutic monitoring of ALS. Key challenges in biomarker development—including undefined pathological neurofilament light chain (NFL) fragments, limited antibody availability, and poor assay reproducibility—are discussed. Strategies, including ultrasensitive detection technologies such as single molecule array (Simoa), antibody optimization based on pathological fragment identification, and multi-omics biomarker panels, should be integrated. These approaches may lead to breakthroughs, pave the way for precision-based ALS diagnosis, provide theoretical support for promoting its clinical translation and application, and offer ideas for future research.