Abstract: The use of Kluyveromyces marxianus NCIM 3465 for the production of ß-D-galactosidase is reported. Experimentation was carried out to optimize the process parameters for maximum enzyme production by varying the parameters such as pH of the medium, temperature, inoculum size, age of inoculum, agitation and incubation time. The maximum enzyme activity was observed with pH 5.0, temperature 30°C, inoculum size 6% (v/v) having 20 h age, under shaking conditions (100 rpm) after 24 h of incubation.