This study investigated the novel application of Rhodotorula rubra as a bio-based collector for selective flotation separation of cassiterite from co-existing minerals, quartz and calcite. Under optimal conditions (Rhodotorula rubra suspension concentration: 2.0g/L, pH=5, and sodium silicate dosage: 3.75 g/L), a cassiterite recovery rate of 65% was achieved, demonstrating effective mineral selectivity. Fourier-transform infrared spectroscopy analysis revealed that the adhesion mechanism between Rhodotorula rubra and cassiterite involved synergistic physical adsorption and chemisorption interactions. These findings provide critical insights into the potential of microbial-based flotation agents for sustainable mineral processing.