Temporal lobe epilepsy (TLE) is the most common epilepsy in both adult and children. Some microRNAs (miRNAs) are abnormally expressed in neurological diseases. This study aimed to investigate the expression level and clinical significance of miR-148a-3p in TLE children and explore its effect on the biological viability of hippocampal neurons. The expression level of miR-148a-3p in the serum of TLE children was examined using quantitative real-time PCR. A receiver operating characteristic curve was plotted to determine the diagnostic accuracy of miR-148a-3p in TLE. Hippocampal neurons were cultured in magnesium-free medium to construct a TLE cell model. The effects of miR-148a-3p on hippocampal neuronal viability and apoptosis rate were detected by MTT and flow cytometry, respectively. miR-148a-3p was overexpressed and correlated with seizure frequency and febrile seizure (FS) history in TLE children. miR-148a-3p was of great value in the diagnosis of TLE, and it can be used to distinguish cases with FS history. Hippocampal neurons treated with magnesium-free medium were used as an in vitro model of TLE and showed significantly increased miR-148a-3p, decreased cell viability, and increased cell apoptosis, while these changes were eliminated markedly by miR-148a-3p knockdown. miR-148a-3p is overexpressed and associated with seizure frequency and FS history and serves as a novel diagnostic biomarker in TLE. In addition, the downregulation of miR-148a-3p exerts neuroprotective role by improving hippocampal neuronal cell viability. miR-148a-3p may provide new ideas for the treatment and diagnosis of TLE.