As a committed step in the urea cycle, arginase cleaves l-arginine to form l-ornithine and urea. l-Ornithine is essential to: cell proliferation, collagen formation and other physiological functions, while the urea cycle itself converts highly toxic ammonia to urea for excretion. Recently, arginase was exploited as an efficient catalyst for the environmentally friendly synthesis of l-ornithine, an abundant nonprotein amino acid that is widely employed as a food supplement and nutrition product. It was also proposed as an arginine-reducing agent in order to treat arginase deficiency and to be a means of depleting arginine to treat arginine auxotrophic tumors. Targeting arginase inhibitors of the arginase/ornithine pathway offers great promise as a therapy for: cardiovascular, central nervous system diseases and cancers with high arginase expression. In this review, recent advances in the characteristics, structure, catalytic mechanism and preparation of arginase were summarized, with a focus being placed on the biotechnical and medical applications of arginase. In particular, perspectives have been presented on the challenges and opportunities for the environmentally friendly utilization of arginase during l-ornithine production and in therapies.