Cold active beta-galactosidases which catalyze lactose hydrolysis and transglycosylation reactions at low temperature make them highly potential biocatalyst in biotechnology, pharmaceutical and food processing industries. Moreover, an interest towards the utilization of diary industrial waste, whey and its constituents, for manufacturing a wide range of valuable products at reliable cost is increasing among researchers in order to facilitate its wider commercial use. In the present study, the fermentation parameters for the maximum production of cold active beta-galactosidase from a psychrotrophic bacterium, Enterobacter ludwigii in cheese whey was optimized by exploring statistical methods, Plackett-Burman design (PBD) and central composite design (CCD). Three most significant factors viz, pH, whey and tryptone out of 11 were selected by PBD and were further optimized by response surface methodology using CCD. The optimal levels of pH, whey and tryptone were indicated as 7.3, 82 (v/v) % and 3.84 g% respectively. An overall 3.6-fold increase in cold active beta-galactosidase production (34.37 U/mL) was achieved in optimized medium compared to the yield from unoptimized medium. The quadratic regression model was proven to be adequate (p = 0.0001, R (2) = 0.9880, CV = 7.96%) and the response (cold active beta-galactosidase production) obtained on validation coincident with the predicted value. Graphical Abstract [GRAPHICS] .