Present investigation was undertaken with an objective to standardize Bio- tea a fermented beverage for suitable substrate and their level of concentration. Cane sugar, Jaggery and Honey were the different substrates used for standardizing the beverage. Among the different substrates tested, beverage prepared using honey as substrate was found to be the best quality beverage in terms of all the sensory parameters with maximum pellicle weight (28.00g), titrable acidity (1.56%), alcohol (2.35%), reducing sugars (13.47 mg/ 100 ml) and Over All Acceptability (OAA) scores (96.86) followed by the beverage prepared using cane sugar with OAA scores 91.07. Sugar was adjudged as the best substrate because of easy availability and economic feasibility when compared with honey. Within the range of substrate concentration studied (50 to 200g per litter of tea decoction), the pellicle weight, pH, Titrable Acidity (T A), alcohol and soluble sugar contents varied significantly affecting the beverage in terms of sensory parameters. The beverage with 10 per cent sugar concentration was highly acceptable, as it attained the desirable much earlier than the beverage with low sugar concentration.