Abstract: Objective To preliminarily investigate the characteristics of serum neurofilament light chain (NfL) and neurophysiological indexes (NI) in amyotrophic lateral sclerosis (ALS), and to explore their auxiliary discriminative value for ALS with different sites of onset.Methods A single-center, cross-sectional exploratory study was conducted. Thirty patients with ALS who were admitted to Department of Neurology, the Affiliated Hospital of Xuzhou Medical University, from September 2024 to March 2026 and met the inclusion and exclusion criteria were enrolled. Meanwhile, 30 age- and sex-matched healthy controls were also included. According to the site of onset, ALS patients were divided into two groups: a bulbar-onset group (n=11) and a spinal-onset group (n=19). Differences in serum NfL levels, NI, and related neuroelectrophysiological parameters were compared between the ALS group and healthy control group, as well as between the bulbar-onset and spinal-onset groups. Binary logistic regression analysis was used to identify factors associated with different onset sites. The discriminative performance of log-transformed NfL lg(NfL), NI, and their combined model for distinguishing ALS with different onset sites was evaluated using receiver operating characteristic (ROC) curve analysis.Results Compared with the healthy control group, the ALS group showed significantly increased serum NfL levels and decreased NI (both P<0.05). The bulbar-onset group had higher NfL levels and NI values than the spinal-onset group (both P<0.05). Logistic regression analysis showed that lg(NfL) and NI were both associated with onset site in the simplified model; however, after additional adjustment for confounding factors, only NI remained statistically significant. The areas under the ROC curve (AUCs) for lg(NfL), NI, and the combined model in distinguishing ALS with different onset sites were 0.758, 0.789, and 0.847, respectively. Although the combined model showed a numerically higher AUC, the difference was not statistically significant compared with either individual indicator.Conclusions NfL and NI may reflect different aspects of ALS pathology, including axonal injury burden and the functional status of specific peripheral motor pathways, respectively. Their combination may provide complementary information; however, the auxiliary discriminative value for ALS with different onset sites requires further validation in larger, multicenter studies.