A total of thirty fungal strains expressing penicillin V acylase activity belonging to genus Aspergillus sp., Fusarium sp. Mucor sp., Penicillium sp. and Rhizopussp. were screened for the production of enzyme under solid state fermentation in conical flasks. Among all fungal strains, Fusariumoxysporum FUS-04 was found to be the most potential strain. β-lactamase test was also performed to get β-lactamase negative strain. Different fermentation media were evaluated for enzyme production. The medium M2 supplemented with wheat bran gave two fold higher enzyme production than M3, M4 and M5. It was observed that the amount of penicillin V acylase produced was highest at substrate to moisture ratio 1:2. Glutamic acid was found to be a best inducer. Concentration and time of addition of glutamic acid was found to be critical for penicillin V acylase production. It was found that production of penicillin V acylase was maximized by mode of addition of inducer. The medium optimization by the Plackett- Burman and central composite designs resulted in six times increase in PVA production.