l-Methioninase is ubiquitous in all organisms except in mammals. It mainly catalyzes the, alpha, gamma-elimination of l-methionine to alpha-ketobutyrate, methanethiol, and ammonia. Unlike normal cells, methionine dependency was reported as a biochemical phenomenon among various types of cancer cells. Thus, l-methioninase is the universal protocol for triggering the majority of tumor cells. This review is an attempt to briefly describe the occurrence of the biochemical and molecular properties of l-methioninase by a comparative manner to the eukaryotic and prokaryotic source for the maximum exploitation in the therapeutic field. The combination of l-methioninase treatment, gene therapy, and chemotherapeutic drugs clearly explores the various therapeutic aspects of this enzyme. Finally, the perspectives for increasing the therapeutic efficacy of this enzyme were described.