The fermentation of an epoxide hydrolase by a newly isolated Trichosporon loubierii strain ECU1040 was optimized. The epoxide hydrolase production was enhanced by 6.9-fold from an initial activity of 45 U/l with the incorporation of substrate induction, optimization of medium composition and other culture conditions, and the implementation of a fed-batch process. It was found that phenyl glycidyl ether (PGE) could efficiently induce biosynthesis of the epoxide hydrolase, though it strongly inhibited the cell growth. To reduce the toxicity of PGE, dibutyl o-phthalate was used in flask cultivation to dissolve PGE, keeping a low concentration of PGE in the broth. Glucose was added to promote cell growth in the presence of PGE, though glycerol was identified as the best single carbon source for the highest specific activity. Trace elements have significant effect on the epoxide hydrolase synthesis.With addition of trace elements and glucose and adjustment of phosphate concentration, the total activity was enhanced by 150%. To further increase the epoxide hydrolase production, a fed-batch culture with PGE in the feed solution was performed in a 5-l jar fermenter. The maximum production of the epoxide hydrolase was 312 U/l, with a specific activity of 23.7 U/g DCW (dry cell weight).