Background: Fermentative production of lactic acid from algae-based carbohydrates devoid of lignin has attracted great attention for its potential as a suitable alternative substrate compared to lignocellulosic biomass. Results: A Chlorella sp. GD mutant with enhanced thermo-tolerance was obtained by mutagenesis using N-methyl-N′-nitro-N-nitrosoguanidine to overcome outdoor high-temperature inhibition and it was used as a feedstock for fermentative lactic acid production. The indoor experiments showed that biomass, reducing sugar content, photosynthetic O2 evolution rate, photosystem II activity (F v/F m and F v′/F m′), and chlorophyll content increased as temperature, light intensity, and CO2 concentration increased. The mutant showed similar DIC affinity and initial slope of photosynthetic light response curve (α) as that of the wild type but had higher dissolved inorganic carbon (DIC) utilization capacity and maximum photosynthesis rate (P max). Moreover, the PSII activity (F v′/F m′) in the mutant remained normal without acclimation process after being transferred to photobioreactor. This suggests that efficient utilization of incident high light and enhanced carbon fixation with its subsequent flux to carbohydrates accumulation in the mutant contributes to higher sugar and biomass productivity under enriched CO2 condition. The mutant was cultured outdoors in a photobioreactor with 6% CO2 aeration in hot summer season in southern Taiwan. The harvested biomass was subjected to separate hydrolysis and fermentation (SHF) for lactic acid production with carbohydrate concentration equivalent to 20 g/L glucose using the lactic acid-producing bacterium Lactobacillus plantarum 23. The conversion rate and yield of lactic acid were 80% and 0.43 g/g Chlorella biomass, respectively. Conclusions: These results demonstrated that the thermo-tolerant Chlorella mutant with high photosynthetic efficiency and biomass productivity under hot outdoor condition is an efficient fermentative feedstock for large-scale lactic acid production.