Nanofibers prepared from cellulose derivatives are a suitable support for enzyme immobilization. We selected a methyl cellulose (MC) nanofibrous mat for enzyme immobilization. First, we established the electrospinning conditions for the preparation of the MC nanofibrous mat. A mixture of ethanol and water at a volume ratio of 1:1 was selected as the solvent of MC for electrospinning. An MC concentration of 5 wt% and a flow rate of 0.5 mL/h were employed to ensure stable electrospinning without bead formation. By varying the applied voltage, the sizes of the MC nanofibers could be controlled in the range of 50 to 80 nm, and an applied voltage of 18 kV was selected for further experimentation. The MC nanofibrous mats were cross-linked with glutaraldehyde under acidic conditions for 12 h in order to increase the stability of the mats in water. Lipase was directly immobilized onto cross-linked MC nanofibrous mats without a further activation step, and 34.82 A mu g of lipase was immobilized per mg of support. The reusability test showed an unexpected loss of activity after second reuse due to a loss of lipase during the washing procedure promoted by the surface erosion of the MC nanofiber. This problem was solved by introducing crosslinked enzyme aggregates (CLEA) to the surfaces of MC nanofibers. CLEA formation enhanced the activity of lipase per mg of support by almost 5-fold compared with the original lipase-immobilized MC nanofibrous mat. The reusability was also enhanced: more than 90% of the initial activity was retained after seven reuses.