To develop a new method for producing aspartame (APM), a simple and efficient strategy for the isolation of certain microorganisms producing APM from soil samples was designed. A newly strain with secreting certain specific dipeptidase to directly synthesize APM from the substrates of L-aspartic acid (L-Asp) and L-phenylalanine methyl ester (L-PM) without protection for amino acid side chains was screened from soil samples. APM concentration in reaction mixture was quantified by high performance liquid chromatography (HPLC), yield reached 0.015 g/L. Examination of the general morphological characteristics and data from the sequence analysis of the rDNA-ITS gene led to identification of the isolate as a strain of fungal endophyte spp. The newly isolated strain had a high potential for application in industrial processes for APM production. In particular, this new method was low cost for synthesis of APM during the reaction due to avoiding protection for amino acid side chains and optical resolution of the mixtures. As we known it, this is first report that a newly strain with a high potential for selective synthesis of the APM was isolated from soil.