The enormous attraction on CO₂ biofixation using photosynthetic microorganisms such as cyanobacteria has been risen due to its promising efficiency and valuable by-products production. In this study, an isolated cyanobacterium from hot spring in Taiwan, Thermosynechococcus sp. CL-1 (TCL-1) was evaluated for its growth factors arrangement effect on the biomass productivity and CO₂ biofixation. The initial biomass concentration, and nutrient supply level variation influenced TCL-1 biomass productivity and CO₂ biofixation rate while the adjusted and controlled pH value gave an insignificant difference on its performance. The initial biomass concentration of 3 g L⁻¹ gave the best result on biomass productivity and CO₂ fixation which reached 143.4 mg L⁻¹ h⁻¹ and 224 mg L⁻¹ h⁻¹ respectively. Regarding to the result of this study, controlled pH value by the CO₂ supply inside the reactor, produced an insignificant difference in TCL-1 performance compared to those with the uncontrolled pH value. The variation of nutrient supply level was achieved by the variation of macronutrient and micronutrient supply inside the medium. The G-solution contains metals and other micronutrient elements which are necessary for the growth of TCL-1. The combination between 5–folds MF medium as the macronutrient, and 3-folds G-solution as the micronutrient supply, present the best TCL-1 performance on biomass productivity and CO2 fixation rate.