Introduction: Pectinase enzyme is one of the most important industrial enzymes which isolated from a wide variety of microorganisms such as bacteria and filamentous fungi. This enzyme has been usually used in the fruit and textile industry. In this study, the isolation and optimization of pectinase-producing fungi on decaying rotten fruits were studied.Materials and methods: Isolation and screening of pectinase producing fungi performed through plate culture on pectin medium and staining with Lugol's iodine solution. The best strains were identified by ITS1, 4 sequencing as Aspergillus fumigatus, Rhizopus oryzae, Penicilium chrysogenum. The enzyme production was optimized by application of the five factorial design, each at three levels. These factors are carbon sources (whey, glucose and stevia), ammonium sulfate, manganese sulfate, temperature, and pH. Pectinase concentration was measured by the Miller method.Results: The results indicate that optimum condition for enzyme production for three fungi strains was obtained at 32°C, pH=6, 3 g/L manganese sulfate, 2.75 g/L of ammonium sulfate and 10 g/L of each carbon source. The best experiment in obtaining the optimum enzyme contained 1.328 mg/ml of glucose for Aspergillus niger 1.284 and 1.039 mg/ml of whey for Rhizopus oryzae and Penicilium chrysogenum. Molecular weight of enzyme was about 40 and 37 kDa which was obtained by SDS- PAGE.Discussion and conclusion: The results indicate that three strains could grow in a wide range of carbon source, pH and temperature, which could be a good candidate for industrial application.