Enantiopure beta-nitroalcohols are versatile intermediates used in the synthesis of important pharmaceuticals and chiral synthons. In this article, immobilizedArabidopsis thalianaHNL (AtHNL)-catalyzed preparation of (S)-beta-nitroalcohols from their racemic mixtures via retro-Henry reaction was studied.AtHNL used in biocatalysis was immobilized by physical adsorption in inexpensive celite (R) 545. Under optimized biocatalytic conditions, the total turnover number of the catalyst has improved 2.3-fold for (S)-2-nitro-1-phenylethanol (NPE) synthesis, than free enzyme catalysis. This study reported for the first time celite-AtHNL-catalyzed retro-Henry reaction at low pH. At pH 4.5 and 5.0, 62% ee and 41% conversion, and 97% ee and 42% conversion of (S)-NPE were obtained respectively, while the free enzyme inactivates at pH < 5.0. The increased catalytic efficiency and pH stability of the catalyst could be possibly due to increased stability ofAtHNL by immobilization. A dozen of racemic beta-nitroalcohols were converted into their corresponding (S)-beta-nitroalcohols using this reaction; among them, eight were not tested earlier. The immobilized enzyme has showed broad substrate selectivity in the retro-Henry reaction, and products were obtained up to 98.5% ee.