Newly discovered strain Baclicus lincheniformis JX010 was identified to enantioselective hydrolysis of racemic ethyl tert-leucinate for the synthesis of chiral L-tert-butyl leucine. In the hydrolysis of isopropyl tert-butyl leucinate, the L-tert-butyl leucine was synthesized in 99% ee and 48% conversion. The cells was immobilized on synthetic resin 0501 without pretreatment to increase the enzyme stability. A series of organic cosolvents were investigated the hydrolysis rate and 2% glycerol was considered as the optimized cosolvent. In the hydrolysis of 50 mM isopropyl tert-butyl leucinate, the immobilized cells remained 85%activity with L-tert-butyl leucine in 99% ee after 8 reaction cycles.