A new method for breeding the hydrocortisone overproducing strain Curvularia lunata by screening ketoconazole-resistance mutant was developed. A hydrocortisone overproducing mutant C. lunata KA-91 with ketoconazole-resistance marker was obtained from protoplasts treated with ultraviolet radiation. The hydrocortisone conversion rate of C. lunata KA-91 was increased by 42.1% compared to the original strain CL-114 at the substrate 17 alpha-hydroxypregn-4-en-3, 20-dione-21-acetate addition concentration of 1.0 g/L. The by-products produced by KA-91 were fewer than those of the original strain. It was assumed that the higher cytochrome P450 content of ketoconazole-resistance mutant resulted in the increase of 11 beta-hydroxylation capacity. The culture conditions for biotransformation of 17 alpha-hydroxypregn-4-en-3, 20-dione-21-acetate to hydrocortisone were optimized by response surface methodology. Plackett-Burman design was applied to elucidate the key factors affecting the hydrocortisone production, and the results indicated that glucose, initial pH, and glucose to total nitrogen sources ratio (omega) had significant effects on hydrocortisone production. Box-Behnken design was employed to search for the optimal parameters of those three key factors. According to the model, the trial checking at the optimal conditions showed a high hydrocortisone conversion rate of 82.67%.