Objectives Multiple sclerosis (MS)-related tremor remains a disabling problem that often responds poorly to medical treatments. This study aims to evaluate the effect of botulinum toxin type A on MS-related upper limb tremor. Methods This retrospective observational cohort study included 8 patients who received intramuscular injections of onabotulinum toxin type A for medically refractory MS-related tremor. Hospital records before and 4–6 weeks after treatment, including the Fahn-Tolosa-Marin Tremor rating scale (FTM-TRS), electrophysiological tremor analysis, neurological examination findings, and videos, were reviewed retrospectively. Results There was significant improvement after botulinum toxin injection in the FTM TRS part A score for tremor (P = 0.011), FTM TRS part B score for hand function (P = 0.011), FTM Part score for activities of daily living (P = 0.012), and FTM TRS total score for overall evaluation (P = 0.012). Electrophysiological tremor analysis revealed maximal tremor amplitudes between 700–2000 μV (before treatment) and between 300–1000 μV (after treatment). The mean tremor amplitudes before and after treatment were 1350 μV and 725 μV, respectively. No adverse reactions were observed after botulinum toxin treatment. Conclusions BoNT may improve upper limb tremor and functionality in MS-related tremor. The treatment involved administering BoNT under EMG guidance, with careful muscle selection based on clinical assessment and EMG findings. This approach aimed to optimize treatment efficacy while minimizing potential adverse effects. Further studies are warranted to confirm these findings.