The lack of energy-sufficient harvesting technologies has dramatically impeded the commercialization of microalgae-derived bio-products. To address this challenge, a foam flotation harvesting approach using bio-surfactants has been previously developed for microalgae. However, most studies have applied chemical surfactants in the foam flotation, which is in opposition to the concept of sustainability. Thus, in this study, two bio-surfactants including rhamnolipid and Bovine Serum Albumin (BSA) were applied in foam flotation-assisted harvesting for Chlorella vulgaris. Initially, the ability of foam formation and foam stability in different microalgal culture media including distilled water, MiracleGro™, and Bold's Basal Medium (BBM) were evaluated. Following this, the microalgal cell adhesion mechanism was elucidated using FTIR analysis, zeta potential, and conductivity. The results showed that a higher degree of foam stability was observed when using BSA as the bio-surfactant than rhamnolipids. No big difference in the zeta potential was observed for BSA in all tested culture media, and the value of zeta potential was approx. 20 mV whereas, a difference was observed between rhamnolipid solution and rhamnolipid in culture media at all tested conditions. On the contrary, in the conductivity analysis, the use of rhamnolipids led to a higher conductivity at all investigated conditions compared to BSA. FTIR analysis suggested that the addition of either rhamnolipids or BSA did not affect the functionality. Overall, this fundamental study regarding the bio-surfactant-assisted foam flotation harvesting strategy for microalgae lays the foundation for process development.