For investigating the influence of coexisting ions (K+, Mg2+, Ca2+ and SO42−), temperature and their synergistic effect on energy generation by reverse electrodialysis, two series of cells dividing with Yadeshi membranes are fed with different solutions at 10 °C, 25 °C and 40 °C. The presence of K+, Mg2+, Ca2+ and SO42− results in lower open circuit voltage, higher internal resistance and lower maximum power density, and the influence order of ions coexisting with NaCl is Ca2+ > Mg2+ (>SO42−) > K+. With the temperature risen, the open circuit voltage and the internal resistance show a trend of increase and decrease respectively, resulting in a bigger power density. Based on the synergistic effect of coexisting ions and temperature, the maximum power density of the pure NaCl system shows a greater increment (0.15 W m−2) than that of NaCl-CaCl2 (0.10 W m−2) and NaCl-MgCl2 (0.11 W m−2) systems when temperature increases from 10 °C to 40 °C. Furthermore, the transport quantities of ions in each system increased with temperature at different degrees, and the uphill of Ca2+ and Mg2+ was more obvious, which can reasonably explain the different effects of temperature on the maximum power density. Moreover, these results are further verified when simulated concentrated seawater is used for both the Yadeshi- and Fujifilm membranes, and the Fujifilm shows better energy generation performance mainly due to a lower internal resistance.