The carboxylesterase gene of Geobacillus stearothermophilus CICC 20156 was cloned by bioinformatics technology and then was inserted into the expression vectors pPIC9K and pYG1.2. The recombined plasmids were transformed into yeast Pichia pastoris GS115 and Aspergillus niger M54 respectively. It was revealed by SDS-PAGE and Western blot that the exogenous protein of about 29 ku with a His-tag each were secreted by the transformed hosts into the external mediums. The concentration of recombinant carboxylesterase in medium excreted by Pichia pastoris and Aspergillus niger was 30.7 mg/L and 15.3 mg/L respectively. Bioactivity assay indicated that the carboxylesterase activity was 22 671 U/mg for the enzyme expressed by Pichia pastoris and 21 438 U/mg for that expressed by Aspergillus niger. The research results also showed that both enzymes expressed by the two kinds of hosts possess similar characteristic. The recombinant enzyme exhibited thermostability with optimum temperature at 60℃ and remained 76.7%～67.6% activity even after incubation at 70℃ for 30 min. The enzyme also showed a broader pH tolerance and the optimum pH for the enzyme activity was at 8.0. This is the first report that the thermostable carboxylesterase of Geobacillus stearothermophilus was highly secretively expressed in Aspergillus niger and Pichia pastoris. Though the amount of carboxylesterase excreted by recombined Aspergillus niger was lower than that excreted by recombined Pichia. pastoris, the engineering Aspergillus niger may still show a better perspective for potential applications in biotechnological industries, for no inducer is needed to get crude carboxylesterases in the culture supernatants of it.