Infection of Human Enterovirus (HEV) containing coxsackievirus has been associated with wide variety clinical illnesses, including aseptic meningitis and central nervous system pathologies. Although diagnosis of HEV infection relies on detection of the virus in the Cerebrospinal fluid (CSF), the virus loads in such specimens tend to be quite low. To improve the sensitivity of HEV diagnosis, the aim of this study was to apply an Immunomagnetic Separation (IMS) technique using affinity polyclonal antibodies against HEV-B to determine the concentration of HEVs in CSF specimens. The difference in the average cycle threshold values between CSF-only samples and CSF samples containing an antibody complex was 1.55. IMS improved the HEV concentration by 3.1-fold, and thus appears to be an effective method for concentrating HEV in the CSF. Therefore, IMS is a useful technique for detecting HEV in the CSF from patients with enteroviral diseases.