The study report a novel esterase obtained from a thermotolerant and halotolerant Bacillus cereus strain AGP-03, isolated from the hot spring of Bakreshwar, West Bengal, India. The enzyme was purified to homogeneity with molecular mass of 41 kDa comprising two polypeptide chains of 25 kDa and 16 kDa with specific activity of 740.17U mg(-1) protein. The enzyme was highly active and stable in wide range of pH (5.5-10.0), temperature (30-80 degrees C) and NaCl concentration (3-11%) with optimum value of 8.5, 55 degrees C and 4.5% NaCl respectively. The enzyme displayed maximum activity toward para-nitrophenyl butyrate (PNPB),K-m and V-max value of the esterase was found to be 52.46 mu M and 1654 U mg(-1) protein respectively against PNPB. Significant inhibition by phenylmethylsulfonyl-fluoride (PMSF) and phenylarsine oxide (PAO) indicated serine and cysteine residues are essential for enzyme catalysis. Moreover, the enzyme exhibited high activity and stability in presence of hydrophobic organic solvent. In presence of benzene the enzyme showed astonishingly high activity (136.9%) and stability. The purified esterase with these outstanding features may have great potential to be used in harsh industrial/biotechnological purposes. Besides, extreme stability in benzene may make this enzyme a potential biocatalyst for the application in non-aqueous based biotechnological processes. (C) 2015 Elsevier Ltd. All rights reserved.