<b><i>Objectives:</i></b> The aim of this study was to explore the frequency of KIR4.1 antibodies in patients with multiple sclerosis (MS) and in control groups using a cell-based assay. <b><i>Materials and Methods:</i></b> A transfected HEK-293A cell line expressing KIR4.1 was established to test for the presence of KIR4.1 antibodies in blood serum. We tested 904 subjects, including 188 patients with MS, 264 patients with neuromyelitis optica spectrum disorders (NMOSD), 209 patients with other inflammatory neurologic disease (OIND), 203 patients with other noninflammatory neurological disease (OND), and 40 healthy controls. <b><i>Results:</i></b> KIR4.1 antibodies were present in 23 of the 188 (12.2%) MS patients, 42 of the 264 (15.9%) NMOSD patients, 32 of the 209 (15.3%) OIND patients, 24 of the 203 (11.8%) OND patients, and 2 of the 40 (5%) healthy controls. There were no significant differences among the MS and control groups (<i>p</i> = 0.279). <b><i>Conclusions:</i></b> Anti-KIR4.1 antibody, as determined by a cell-based assay, is not a specific biomarker for MS.