Pyrene is a polycyclic aromatic hydrocarbon (PAH) commonly observed in aquatic ecosystems, which originates primarily from the incomplete combustion of fossil fuels and the use of petroleum compounds. Pyrene can cause the immune disturbance and oxidative stress, result in immunotoxicity, DNA damage, reduce reproduction significantly, and induce behavioral changes. Marine bivalves are commonly used as bioindicators for marine pollution, and hemolymph is a metabolite transfer medium for PAH pollutant. However, the vital immune indicator responses of pearl oyster Pinctada martensii hemolymph exposed to pyrene is still unclear. Thus, the immunotoxic responses of pyrene on the hemolymph of the Pinctada martensii were investigated in this study. After exposure to pyrene for 7 days, the total number of hemocytes (THC), cell membrane stability (CMS), phagocytic activity (PA) and total glutathione (GSHT) all decreased significantly. Pyrene also caused a significant increase in lipid peroxidation (LPO). Median effective concentrations (EC50) of pyrene on THC (4.5 μg L−1) and LPO (5.2 μg L−1) were lower than those for CMS (13.8 μg L−1), PA (12.1 μg L−1) and GSHT (7.2 μg L−1), which indicates that THC and LPO were more sensitive. Additionally, a clear dose-effect relationship indicated that pyrene stimulated a marked immune response, as well as oxidative stress in P. martensii, which demonstrates the subtle effects of pyrene exposure on marine invertebrates and the potential associated risk.