This study aimed to figure out the optimum fermentation temperature by analyzing the major chemical constituents of black tea and its antioxidant activities, inhibitory activities against α-amylase, α-glucosidase and glucose uptake. Sensory evaluation revealed 28°C-fermented black tea showed best sensory quality with highest scores for aroma (6.5) and taste (29.8). With increasing fermentation temperature, polyphenols, polysaccharides, (−)-epigallocatechin, (+)-catechin, theaflavins and thearubigins decreased significantly, while amino acids, soluble sugars and volatiles increased when temperature increased up to 31 °C. Furthermore, black tea fermented at 28 °C showed strongest DPPH radical scavenging ability, anti-lipid peroxidation and inhibitory effect on α-glucosidase activity with IC50 value of 17.72 μg/mL, 1.49 mg/mL and 21.69 μg/mL respectively. While 25°C-fermented black tea showed highest inhibition rate of glucose uptake in Caco-2 cells. The results demonstrate that appropriately lowering fermentation temperature could improve both sensory quality and bioactivity of black tea.