l-Ornithine, a non-protein amino acid, is widely used as a food supplement and nutrition product that is usually produced by the enzymatic hydrolysis of l-arginine. However, urea, a by-product, complicates purification of l-ornithine. Here, we describe a highly efficient coupled system, which involves Rummeliibacillus pycnus arginase and Jack bean urease, for the production of l-ornithine without urea residue. Although R. pycnus arginase exhibited the highest enzyme activity at pH 9.5 with Mn2+, it could be activated by Ni2+ at 70°C and pH 6.5. Kinetic study showed that, compared with Mn2+-containing arginase, Ni2+-containing arginase had a higher substrate affinity and catalysis efficiency. Based on the properties of commercial Jack bean urease and recombinant arginase, both enzymes showed robust activity at pH 6.5 and 40–50°C. Using a coupled system involving arginase and urease for bioconversion, l-ornithine production of 37.8gl−1 was achieved with a 99.7% molar yield within 5h. Thus, this coupled system represents an applicable strategy for the biosynthesis of l-ornithine.