Objective. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with unknown causes involving upper and lower motor neurons. Our study aimed to explore the diagnostic value of neuroelectrophysiological examination in patients with amyotrophic lateral sclerosis. Methods. A total of 64 patients admitted to our hospital from January 2014 to December 2020 were selected as ALS group. Additionally, 64 normal healthy people in the same period were selected as the control group. Electrophysiological tests were performed on all personnel involved in the study, and the results and parameter changes of different personnel were compared and analyzed. Results. There was a statistical difference between the EMG data of clinically confirmed ALS and the proposed ALS and possible ALS ( p < 0.05 ). The abnormality of confirmed ALS was particularly obvious, and the overall abnormal rate of EMG in ALS was 85%. The CMCT of the upper and lower limbs of clinically diagnosed ALS, suspected ALS, and possible ALS were longer than that of the normal group ( p < 0.05 ). There was no significant statistical difference between clinically diagnosed ALS and suspected ALS ( p > 0.05 ), and there was a difference between clinically diagnosed ALS and possible ALS ( p < 0.05 ). In ALS group, the frequency of F wave decreased, which may be related to the involvement of F wave conduction pathway and the degree of lower motor neuron involvement ( p < 0.05 ). In addition, the amplitudes of F and H waves increased, which was related to the involvement of upper motor neurons ( p < 0.05 ). In ALS group, SCV was hardly involved, and CMAP decreased significantly, which was positively correlated with the degree of muscular atrophy and muscle strength decline ( p < 0.05 ). The sensitivity of electrophysiological detection was 76.56%, the specificity was 78.33%, and the AUC was 0.8578. Conclusion. Patients with clinically suspected ALS should undergo electrophysiological testing as soon as possible, which is conducive to the early diagnosis and differential diagnosis of ALS. This trial is registered with ChiCTR2100046535.