We aimed to develop microbial production systems for lactobionic acid (LA) for utilization of surplus lactose (Lac) from whey. Screened and selected strain Burkholderia cepacia No. 24 grew even in highly concentrated Lac syrup and had high oxidizing activity against Lac. We examined the conditions for fermentation and microbial conversion of Lac to LA. A three-day batch culture produced 200 g/L of LA. A ten-day fed-batch culture with periodic additions of Lac and CaCO₃ accumulated 400 g/L of LA. Lac (150 g/L) was completely converted to LA by bioconversion with Burkholderia cepacia cells in 24 h.