Kombucha is produced by the action of sugar and fermentative symbiotic colonies of bacteria and yeast (SCOBY) in black and green tea infusions. The main active substances in kombucha are polyphenol compounds (epigallocatechin gallate, epicatechin gallate, gallocatechin gallate, epicatechin, epigallocatechin, gallocatechin, and catechin), vitamins (B1, B2, B6, B12, and C), and organic acids (D-gluconic acid, and glucuronic acid). Several studies have reported that the main health benefits of kombucha include its energizing and detoxifying properties, in addition to anticancer and anti-aging effects. Sugar is a very important component necessary for glucose fermentation. However, this may result in increased consumption of calories depending on the sugar content. Therefore, this study investigated the quality characteristics of commercial kombucha and manufactured low-calorie kombucha (MLK) using alternative sweeteners (non-digestible maltodextrin, enzymatically modified stevia, or sucralose) and different levels of sugarcane content. The total soluble solids content, pH, color, organic acids, catechins, and caffeine content in kombucha during fermentation were then measured. The results showed that there was no significant difference in color. However, the pH of the MLK decreased from 5.4 to 2.81 during fermentation. Interestingly, the highest D-gluconic acid content was observed when 5% (w/v) sugarcane was added to the MLK and fermented for 16 days. The catechin contents did not change during fermentation. The caffeine content decreased from 2.00 ± 0.01 mg/100 mL to 0.94 ± 0.00 mg/100 mL. In conclusion, this study developed low-calorie kombucha using alternative sweeteners, which suggests a new trend in the manufacture of kombucha.