Psoralen, an important furanocoumarin occurring abundantly in seeds of Psoralea corylifolia is used as an anticancerous compound against leukemia and other cancer cell lines. Evaluation and isolation of psoralen from the calluses derived from different plant parts, viz. cotyledons, nodes, leaves and roots have been done in the present case for the first time. Amongst all, a maximum of 1934.75 μg/g f.w. of psoralen was recorded in callus derived from cotyledons, followed by 1875.50 and 1465.75 μg/g f.w. of psoralen in node and leaf derived calluses, respectively. Amount of psoralen enhanced further when cotyledonary calluses were exposed to different concentrations of organic elicitors (yeast extract, proline, inositol, casein hydrolyzate (CH), glycine, glutamine and sucrose) and precursors of psoralen (umbelliferone, cinnamic acid and NADPH). Isolation of psoralen was done using methanol as solvent through column chromatography and TLC. FT-IR and NMR further characterized and confirmed the structure of psoralen. In addition, the putative gene, psoralen synthase involved in psoralen synthesis pathway has been isolated, cloned and sequenced which comprised 1237 bp length. BLAST analysis of the gene sequence of psoralen synthase revealed that its nucleotide sequence showed 93% homology with psoralen synthase isolated from Ammi majus. This is the first report of isolation, cloning and characterization of psoralen synthase from Psoralea corylifolia.