The gene encoding extracellular thermostable carboxylesterase (estA) from Geobacillus thermoleovorans YN was fused to six-histidine and expressed under different promoters. Three different clones carrying this gene were constructed in host cell Escherichia coli BL21 (DE2) using three expression systems pET-17b, pBluescript ® II KS(+) and pCYTEX-P1 (pET-estA T7- promoter; pBlue-estA T7-promoter and pBlue-estA T7 promoter & Lambda pL promoter). The efficiency of expression of the three constructs was evaluated, where the highest esterase expression (589 u/ ml) using p-Nitrophenyl laurate as substrate (pNP-laurate: C18H27NO4) was measured under the control of T7-promoter in expression vector pET-17b after 4 h of the induction. A 1.5 fold increase in enzyme activity was measured with specific activity 1043 u/ mg protein on growing the clone expressed the target gene under T7-promoter (pET-estAT7) in 2-L working volume BIOFLO III bioreactor under optimal conditions. Recombinant expression of the tested thermostable esterase was monitored by sodium dodecyl sulphate polyacrylamid gel electrophoresis, zymogram and activity measurements with ?--Naphthyl acetate (C12H10O2) and Fast Red. The SDS-PAGE analysis of E. coli BL21(DE3)/pET-estA lysates indicated the presence of a protein band (29 kDa) related to the targeted expressed gene.