Nitrogen availability directly affects the microalgal metabolism. A thermophilic cyanobacterium named Thermosynechococcus CL-1 was cultivated in a continuous system to evaluate the effects of NO3– fluxes on the biomass production, bioenergy production, and CO2 fixation. The results show that decreasing the NO3– flux to a N-deprived level (1.01 mM/d) enhances the carbohydrate content in TCL-1 to 45%, accompanied by a decrease in the lipid content. However, increasing the NO3– flux from a N-deprived level (1.01 mM/d) decreases the carbohydrate content dramatically, accompanied by a slight increase in the lipid content. No matter whether the NO3– flux decreases from a N-replete level (8.35 mM/d) to a N-deprived one (1.01 mM/d), or increases from a N-deprived level to higher one, the peak biomass yield occurs at the same NO3– flux level, 4.18 mM/d. In addition, the peak lipid yield, carbohydrate yield, and CO2 fixation rate were recorded at 482 and 660 mg/L/d, and 3.9 g/L/d, respectively, under the same NO3– flux level (4.18 mM/d). Although cultivating TCL-1 under the 4.18 mM/d NO3– flux level exhibits great biomass production, CO2 fixation, and bioenergy production potential, different procedures (the NO3– flux decreasing from the N-replete level and the NO3– flux increasing from the N-deprived level) could influence the final quantity of bioenergy production and CO2 fixation. The NO3– flux, NO3– concentration in the bioreactor, and the NO3– flux variation routes are all important factors to determine the nitrogen availability of TCL-1 in continuous cultivation.