Exposure to severe stress can lead to the development of neuropsychiatric disorders, including post-traumatic stress disorder (PTSD). The cause of PTSD is dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis and an imbalance of monoamines. Fruits and vegetables contain large amounts of luteolin (LU; 3′,4′,5,7-tetrahydroxylflavone), which has various pharmacological activities such as anti-inflammatory, antioxidant, and anti-allergic effects. We investigated the effects of LU on fear, depression, and anxiety following monoamine imbalance and hyperactivation of the HPA axis in rats exposed to single prolonged stress (SPS). Male rats were dosed with LU (10 and 20 mg/kg) once daily for 14 days after exposure to SPS. Administration of LU reduced fear freezing responses to extinction recall and depression- and anxiety-like behaviors, and suppressed increases in plasma corticosterone and adrenocorticotropic hormone levels. Also, administration of LU restored the increased norepinephrine and decreased serotonin levels in the structures within the fear circuit, medial prefrontal cortex, and hippocampus. Our results showed that administration of LU improved freezing behavior according in a situation-dependent manner, and showed anti-depressant and anxiolytic effects. Thus, LU may be a useful therapeutic agent to prevent traumatic stress such as PTSD.