Cutinases are hydrolytic enzymes which catalyzes esterification and trans-esterification reactions that make them highly potential industrial biocatalyst. In the present investigation microorganisms showing cutinase activity were isolated from plant samples. The strain showing maximum cutinase activity was identified by 18S rDNA sequencing as Aspergillus sp. RL2Ct and was selected for further studies. To achieve maximum enzyme production, the medium components affecting cutinase production were screened by Plackett-Burman followed by central composite design. The results obtained suggested that cutin, temperature and CaCl2 have influenced the cutinase production significantly with very high confidence levels. Cutinase production was maximum (663 U/mg protein) when using cutin prepared from orange peel as sole source of carbon. An overall 4.33-fold increase in the production of cutinase was observed after optimization of culture conditions (including 2.5-fold increase using RSM) during 24 h of incubation. The production time of Aspergillus sp. RL2Ct cutinase is significantly lower than the most of the earlier reported cutinase-producing fungus.