Much endeavor has been dispensed recently to evaluate the potential of CO mitigation by microalgae. We introduce an alternative, novel, LED-illumination isothermal microcalorimetric method to assess the thermodynamic behaviors of microalgae for better understanding of their carbon sequestration capacity. Microalgae thermodynamic behaviors were recorded as power-time curves, and their indices such as total heat evolution (Q), maximum power output (P) and heat generated by per algae cell (J) were obtained. The values for highest (74.80 g L) and control sample (0.00 g L) of Q, P and J were 20.85 and 2.32 J; 252.17 and 57.67 μW; 7.91 × and 8.80 × J cell, respectively. According to the values of Q, a general order to promote the CO sequestration was found at 74.8 g L > 29.92 g L > 14.96 g L > 7.48 g L > 0 g L of C sources, which directly corresponded to carbon availability in the growth medium. Chlorella vulgaris GIEC-179 showed the highest peak P at 74.8 g L concentration which was directly transformed to their biomass during bio-fixation of CO process. This study is applicable for better understanding of CO fixation performance of algae.