This paper evaluates the potential of Chlorella sp. microalgae biomass as a feedstock for biosuccinic acid (bio-SA) production via the fermentation process. In this study, the biomass was pretreated and hydrolyzed using a mild acid before being fermented with Actinobacillus succinogenes. The influence of the initial biomass concentration and agitation rate on bio-SA production during batch fermentation was evaluated using a Response Surface Methodology (RSM) design. The results of the study indicated that Chlorella sp. microalgae biomass contained various types of sugars, with glucose identified as the dominant reducing sugar in the Chlorella sp. hydrolysate. According to the RSM analysis, the study showed that changes in the initial biomass concentration and agitation rate could significantly affect bio-SA production from Chlorella sp. hydrolysate. The highest bio-SA concentration of 14.56 g/L with a yield of 0.62 g/g was achieved when fermentation was performed using 10% (w/v) biomass at 150 rpm. Therefore, this study suggests that Chlorella sp. hydrolysate can be an alternative and renewable feedstock for efficient bio-SA production.