L-lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to L-lactate using NADH as a cofactor. In this work, we have optimized the immobilization of LDH from rabbit muscle in glyoxyl-agarose to have an active and stable preparation which is able to synthesize L-lactic acid. Optimization of various parameters during immobilization allowed the preparation of an active and highly stable immobilized derivative of LDH. A 90.1% immobilization and 72.0% yield was achieved using 300 mM trehalose during the immobilization process. Thermal stabilization factors attained for the immobilized LDH were 1600 times greater as compared to its soluble counterpart. The immobilized preparation was also stabilized against ethanol where it recovered 75% of its initial activity after 48 h while the soluble enzyme was completely inactivated after only 10 min under the same conditions. The enzyme activity profile versus pH and temperature showed a wider range for the immobilized preparation as compared to the soluble enzyme, whilst maintaining optimal values. Production of L-lactic acid was achieved in a batch reactor with the immobilized LDH and this preparation resisted 15 reuses without the loss of activity. We believe that the immobilization strategy optimized here broadened the possibilities of utilizing LDH for biocatalytic applications. (C) 2016 Elsevier Ltd. All rights reserved.