Objective An ideal animal model of epilepsy can be used to study the pathogenesis of epilepsy and evaluate novel antiepileptic drugs. To optimize the simulation of the pathological characteristics and seizure behavior of medial temporal lobe epilepsy (MTLE), we aimed to establish a chronic epilepsy model of MTLE by unilateral, single hippocampal injection of kainic acid (KA) via stereotactic surgery, and to validate this epilepsy model in terms of behavior, electrophysiology, and pathology. Methods A total of 22 healthy C57BL/6 wild-type male mice were divided randomly into a control group (n = 6) and an experimental group, which received KA injections (n = 16). Both groups underwent microinjection of equal doses of saline or KA in the hippocampal CA3 area via stereotactic surgery. One week later, all mice were implanted with electrodes in the hippocampal CA3 area to facilitate electroencephalogram (EEG) recording. Seizure frequency and duration were analyzed statistically. The chronic epilepsy model was assessed in terms of behavior, electrophysiology, and pathology. Results No mice in the control group experienced seizures, while all surviving mice in the experimental group developed seizures. Adult model mice exhibited chronic spontaneous seizure behaviors, such as staring, chewing, head and facial muscle twitching, and limb spasms. Two mice died as a result of the surgery, four mice died during the acute seizure phase, and 10 model mice were successfully established. EEG recordings showed epileptiform changes consistent with MTLE. Immunofluorescence staining revealed neuronal loss in the CA3 area and astrocytic changes, consistent with characteristic pathological changes of hippocampal sclerosis. Conclusions We successfully established a chronic epilepsy model of MTLE by unilateral, single intracranial injection of KA and confirmed the validity of the model in terms of behavior, electrophysiology, and pathology. This KA model possesses several advantages, including being time-efficient, easy to perform, cost-effective in terms of drug usage, easily replicated, and being KA-dose-dependent. This epilepsy model exhibits similarities to human MTLE in terms of EEG, behavior, and neuropathological changes, and can thus be used to study effective drugs for treating temporal lobe epilepsy and as an ideal animal model for predicting the outcome of epilepsy surgery.