Kombucha is a beverage obtained by the fermentation of sugared tea with tea fungus. The tea fungus converts the sweetened tea to a refreshing beverage with a variety of beneficial micronutrients. The presence of heavy metals in beverages is a major concern for public health due to their biotoxic effects. In this work, the heavy metal removal potential of live tea fungus from Kombucha drink is studied. The effect of four influencing parameters on biosorption of metals, including initial tea fungus dosage, tea content, sugar content and water hardness, was investigated and optimized by central composite design (CCD) under response surface methodology (RSM). The analysis of variance (ANOVA) of quadratic models demonstrated that all models were statistically significant (p-value < 0.0001). The optimum conditions were found to be 35.85 (g), 4.43 (g), 18.42 (g) per 500 mL of the drink, for initial tea fungus dosage, tea content, sugar content, respectively and 0.0 (mg/L) for water hardness. Under these optimized conditions, the experimental removal efficiencies for Hg2+, As3+, Pb2+, Cd2+, and Cr6+ were 93.3%, 76.7%, 76.1%, 84.3% and 75.4%, respectively. The experimental values of optimum biosorption showed an appropriate agreement with the predicted values of CCD.