Cheese whey (CW) is the liquid by-product of cheese and yogurt making. This potential pollutant has high-quality nutrients exploitable through fermentation processes. Using high-intensity ultrasound on dairy products has shown several technological advantages for bioprocesses. Therefore, this study aimed to investigate the effect of high-intensity ultrasound (HIUS) on kefir grains biomass increase and specific metabolites in CW kefir. Fresh CW was ultrasonicated at 9.0 ± 2.7 and 18.0 ± 3.0 W/cm2 for 30 and 180 s, inoculated with kefir grains, and fermented for 40 h. Total exopolysaccharide production, kefir grains biomass increase, titratable acidity, pH, and soluble solids were analyzed every 8 h. CW pretreated with 18.0 ± 3.0 W/cm2 for 180 s and fermented for 16 h had significantly higher (p < 0.05) total exopolysaccharide concentration than the control: 212.7 ± 0.0 and 186.6 ± 0.0 mg/L, respectively. Ultrasonicated CW at 18 W/cm2 for 30 and 180 s at 24 h fermentation time had significantly higher kefir grains biomass (p < 0.05) than the control: 44.2 ± 0.8 and 43.6 ± 0.9 g/L, and 40.5 ± 0.4 g/L, respectively. Fresh CW pretreated with HIUS enhanced the biosynthesis of kefir beverage total exopolysaccharides concentration and kefir grains biomass.