Stevia rebaudiana is a vital medicinal plant of the genus Stevia and family Asteraceae. It is commonly used as a natural sweetener plant and its products are 300 times sweeter than the commonly used sugarcane. The sweetening potential is due to the presence of calorie-free steviol glycosides (SGs). The plant species has been extensively profiled to identify steviol glycosides (SGs) with intensity sweetening properties. However, the limited production of plant material is not fulfilling the higher market demand worldwide. Researchers are working worldwide to enhance the production of important SGs through the intervention of different biotechnological approaches in S. rebaudiana. In this review, the research work conducted in the last twenty years, on the different aspects of biotechnology to enhance the production of SGs has been precisely reviewed. Biotechnological methods such as micropropagation, callus and cell cultures, elicitation and the metabolomics and transcriptomic elucidation of the biosynthetic metabolic pathways for the production of steviol glycosides have been concisely reviewed and discussed.