The present work emphasizes on citric acid production from distillery spent wash containing carbohydrates that has been tested to be novel and potential substrate. Pre-treatment of distillery spent wash was studied in an anaerobic digestion by suspended growth system under shifting hydraulic retention time and organic loading rate as well as physicochemical parameters. Fungi strain PN12 was applied for citric acid production using raw post methanated waste (treated spent wash). A potent fungus was identified by molecular characterization (18S rRNA). To find phylogenetic relatedness gene sequence of potent culture PN12 has been compared with other sp. gene sequences registered at database of NCBI and the phylogenetic tree was created by software. For enhancement of citric acid production was carried out in different nutritional and environmental parameters such as inoculum size (2 to 10 %), incubation period (24 to 144 h), pH (2 to 7), temperature (25 to 37°C), nitrogen source (organic and inorganic 0.1 g/L), phosphorus source (0.1g/L). The detection of enzymes activities specified that the pathway of pyruvate carboxylation enhanced, which proposed carbon flux to citric acid it was reorganized in strains. Analysis of biosynthesized products by UV/Vis spectrophotometer and HPLC. Initial sugar and consumed sugar determined by DNSA method and also estimated cell dry weight.