Probiotic vegetable-based beverages, such as lychee, can be rich in nutrients, free of cholesterol and lactose, and also have few allergenic components. Saccharomyces boulardii is an alternative to make lychee juice a probiotic beverage. This work aimed to develop probiotic lychee beverage (LB) using S. boulardii by evaluating the effect of refrigerated storage on cell viability, physicochemical characteristics, and acceptance. LB supplemented with S. boulardii was fermented (24 h/30°C), supplemented with sucrose (4-12 °Brix), and refrigerated (up to 28 days/4°C). The yeast viability, total soluble solid (TSS) and, ethanol content, pH, total phenolic compounds (TPC), and AA levels were evaluated over 28 days of storage. Also, the profiles of sugars, organic acids, and phenolic were determined by HPLC/UHPLC. The sensory acceptance of the probiotic beverage was evaluated. Higher sucrose levels resulted in greater yeast viability (3,9 log CFU/mL) on the 21st day of storage and superior TPC and ethanol levels. Storage reduced the TPC, AA, and TSS. LB supplemented with sucrose, probioticated by S. boulardii, and stored for 21 days became accepted by the consumer and presented an adequate physicochemical profile with probiotic potential (> 3,9 log CFU/mL). Probiotication of lychee beverage is an alternative to dairy-based probiotic beverages.