Polyclonal antibodies against the 68 kDa esterase ofFusarium sporotrichioideswere produced by immunizing rabbits with the enzyme, which had been partially purified by ammonium sulfate precipitation, DEAE‐Sephadex anion exchange chromatography and gel electroelution. The antiserum was further purified by passage through an affinity column armed with proteins obtained from the affinity chromatography step, having immunoreactivity but containing no enzyme activity. Immunoblot analysis revealed that the antibodies obtained after the subtractive affinity chromatography purification reacted primarily with one protein band corresponding to esterase type III, at around 68 KDa, and some other minor protein bands when large amounts of the crude extracts were used. Using the purified antibodies, an indirect ELISA for the quantification of esterase type III was established. A good correlation (r = 0.8P =0.04) was found between the ELISA data of esterase III amount and the enzyme activities in the crude extracts. The increased activities of fungal esterase with the prolonged incubation from 5 to 35 days were found to be due to an increase in esterase production. High temperature (25°C) was preferred for the enzyme production and enhanced esterase activity.