Sacrosidase (invertase) is a glycoside hydrolase that catalyzes the hydrolysis of sucrose into glucose and fructose. Sacrosidase is naturally produced in the human small intestine. In contrast, microbial sacrosidase primarily from yeasts (e.g., Saccharomyces cerevisiae), fungi (e.g., Aspergillus niger), and certain bacteria offers scalable production through fermentation for both industrial and therapeutic applications. Initially valued in the food and beverage industry for invert sugar production, sacrosidase has evolved into a clinically relevant enzyme for treating congenital sucrase-isomaltase deficiency (CSID). Production strategies include submerged fermentation (SmF) and solid-state fermentation (SSF), with SSF often yielding higher enzyme titers for specific strains. Advances in statistical optimization methods, recombinant DNA technology, and genetic engineering have significantly enhanced enzyme yield, purity, and stability. Downstream processes such as membrane filtration and immobilization further improve industrial feasibility by increasing operational stability and reusability. Invertase also exhibits several health benefits, with emerging applications in functional foods, nutraceuticals, and biopharmaceuticals. This chapter integrates biochemical and applied perspectives, highlighting the transformation of microbial sacrosidase from a historical biochemical curiosity into a versatile, high-value biocatalyst with enduring significance in both health and industry.