Increased ocean temperature due to global warming affects the health and immunity of fish. In this study, juvenile Paralichthys olivaceus were exposed to high temperature after pre-heat (Acute: Acute heat shock at 32 °C, AH-S: Acquired heat shock at 28 °C & short recovery (2 h) and heat shock at 32 °C, AH-L: acquired heat shock at 28 °C and long recovery (2 days), AH-LS: acquired heat shock at 28 °C & long (2 days) + short (2 h) recovery). Heat shock after pre-heat significantly upregulated various immune-related genes, including interleukin 8 (IL-8), c-type lysozyme (c-lys), immunoglobulin M (IgM), Toll-like receptor 3 (tlr3), major histocompatibility complex IIα (mhcIIα) and cluster of differentiation 8α (cd8α) in the liver and brain of P. olivaceus. This study showed pre-exposure to high temperatures below the critical temperature can activate fish immunity and increase tolerance to high temperatures.