Enterovirus (EV) belongs to the picornavirus family and is common pathogen in clinics. Early study regarding EV infection mainly utilized pathogen culture, but retarding diagnosis and treatment. Recent progress of molecular biology has made progress for pathogen diagnosis. This study thus investigated the expression of EV in cerebrospinal fluid (CSF) of children with central nervous infection, in an attempt to discuss the clinical significance of EV genotyping. 94 children with aseptic encephalitis (or meningitis) were enrolled in parallel with 37 controls. Fluorescent RT-PCR was used to screen EV from CSF, with positive results obtained from those samples with CT values less than 30. After primary screening of EV, amplification of VP1 and VP4 segment was then performed, followed by sequencing of positive fragments and genotyping. No EV RNA was detected in 37 control patients, while 69 out of 94 aseptic encephalitis patients had EV RNA expression. Among those 53 samples were positive for VP1 segment while 47 samples were VP4-positive. Sequencing of those segments showed more than 96% homology with Gen Bank EV standard viral strain. Fluorescent RT-PCR can detect EV RNA from CSF of central nervous infection children with efficiency and accuracy. The combined genotyping of VP4 sequence can further improve sensitivity of EV typing.