Background: levo-Menthol is an important flavoring chemical, which can be prepared by enantioselective enzymatic hydrolysis of dl-menthyl esters. A recombinant esterase (BsE) cloned from Bacillus subtilis 0554 shows excellent enantioselectivity to dl-menthyl acetate and has been immobilized using cross-linked enzyme aggregates. Though BsE has relatively high substrate tolerance, the conversion of dl-menthyl acetate decreased sharply with the increase of substrate loading from 1 to 3 M in mono-aqueous system, which might be due to the severe inhibition of enzyme activity at extremely high load of substrate or product. In this work, enzymatic hydrolysis of dl-menthyl acetate with an extremely high load using the immobilized CLEA-BsE was investigated in an organic-aqueous biphasic system containing surfactant to establish a promising bioprocess for large-scale production of l-menthol. Results: An efficient biphasic reaction system of pentanol-water containing sodium dodecyl sulfate (SDS) was developed for improving enantioselective hydrolysis of dl-menthyl acetate to produce l-menthol by immobilized BsE. Under the optimized reaction conditions, l-menthol was produced in >97% enantiomeric excess (ee) at a substrate load of up to 3.0 M with >40% conversion. Conclusions: All the positive features demonstrate the potential applicability of the bioprocess for the large-scale production of l-menthol.