Roots of Asparagus racemosus were used as a source of inulin for the production of inulinase from Kluyveromyces marxianus YS-1. Root extract prepared at 10 kg/cm2 for 10 min showed the maximum production of inulinase. Various parameters of fermentation have been examined in order to improve overall enzyme yield. Inulinase yield of 40.5 IU/mL in an optimized medium containing inulin (3.5%), beef extract (0.5%), MnSO4 (0.5 mM), CoCl2 (0.05 mM), SDS (0.4 mM) and pH 6.5 at 30 °C under agitation (150 rpm) for 60 h has been obtained at shake flask level. After optimization the enzyme production has been increased 6.1 times at flask level. In a bioreactor (working volume 1 L) fermentation medium containing 4% inulin has shown maximum inulinase production (47.3 IU/mL) under optimized parameters of 200 rpm, 0.75 vvm aeration at 30 °C after 60 h.