The study aimed to investigate the effects of temperature and dissolved oxygen on juvenile largemouth bass during transportation. The experiment involved four temperature groups: 20, 15, 10, and 5 °C. We analyzed the effects of acute and uniform cooling on fish behavior to determine the optimal approach for cooling. Then, we simulated transport under different temperature conditions while measuring the dissolved oxygen level and metabolic rate until all the fish died. The results showed that acute cooling significantly influenced the tail-beat frequency of fish compared with uniform cooling, while abnormal behaviors such as increased swimming, attempted jumping out of the water, and loss of balance were observed. As the transport temperature reduced, the oxygen consumption rate of fish significantly changed at 10 °C, being 2.6 times lower than at 15 °C, with values of 0.10 ± 0.02 and 0.47 ± 0.07 mg·g⁻¹·h⁻¹, respectively. The critical oxygen threshold (P_crit) of fish were 1.90 ± 0.12, 1.61 ± 0.04, 1.15 ± 0.09, and 1.12 ± 0.25 mg·L⁻¹ at 5, 10, 15, and 20 °C. In addition, below P_crit, hypoxia-led behavior changes and oxygen consumption rate reduction were observed at every transport temperature. The findings suggest that the optimal low temperature can reduce metabolism and improve the hypoxia tolerance of juvenile largemouth bass. We recommend transporting largemouth bass at an optimal low temperature (15 °C), monitoring fish behavior, and maintaining oxygen levels above P_crit during transport to prevent stress.