Cibacron Blue F3GA (CB) dye and lipase from Candida rugosa were assembled into multiple alternating bilayers on ultra-fine cellulose (Cell) fiber surfaces via electrostatic layer-by-layer (LBL) deposition. The presence and even coverage of CB and lipase molecules on the fibers were clearly evident by FTIR spectra and microprobe element mapping. The average thickness of each CB/lipase bilayer was similar to 11 nm and the total LBL thickness increased with increasing numbers of bilayers up to 5 bilayers. The CB and lipase loadings on the Cell fibrous membrane also increased with the increasing bilayers. While CB was loaded at similar to 8.1 mg/g Cell from layers 2-4, lipase loading was similar to 3.3 mg/g Cell for the first 2 then became more varied, i.e., between 1.5 and 6.5 mg/g Cell, from layers 3-5. The maximum catalytic activities of lipase bound in the CB/lipase bilayers were 97.0, 90.4, 85.2, 76.1, and 55.6 U/mg of lipase with 1-5 bilayers, respectively. The catalytic activity of lipase bound in all 5-bilayer LBL was equivalent to 45% of that of free lipase. The successful assembling of alternating layers of CB and lipase as shown by incrementally increasing thickness and loading demonstrate that LBL is a promising approach to immobilize enzymes as nano-films on 3D fibrous templates. (C) 2009 Elsevier B.V. All rights reserved.