Low temperature is an important environmental factor in aquaculture farming that affects the survival and growth of organisms. In the present study, we investigated the effects of low temperature on biochemical parameters, oxidative stress and apoptosis in pufferfish. In the stress group, water temperature decreased from 25 °C to 13 °C at a rate of 1 °C/1 h. Fish blood and liver were collected to assay biochemical parameters, oxidative stress and expression of genes at 25 °C, 21 °C, 17 °C, 13 °C and 13 °C for 24 h. The results showed that low temperature could decrease total blood cell count, inhibit cell viability, and subsequently lead to DNA damage. Biochemical parameters such as plasma protein and ALP significantly declined in fish under low temperature, while a significant increase in AST, ALT, LDH and glucose was observed. The gene expression of antioxidant enzymes (SOD and CAT), HSP90 and C3 were induced by low temperature stress. Furthermore, the gene expression of apoptotic related genes including P53, caspase-9 and caspase-3 were up-regulated, suggesting that caspase-dependent pathway could play important roles in low temperature-induced apoptosis in fish. This study may provide baseline information about how cold stress affects the physiological responses and apoptosis in fish.