β-N-acetylglucosaminidases (NAGases) participate in the removal of N-acetylglucosamine (GlcNAc) from the non-reducing end of peptidoglycan or chitin, and are important for many biological functions and industrial applications. [Objective] A new NAGase encoding gene NagZ703 was cloned from a facultative alkaliphilic Bacillus pseudofirmus and expressed in Escherichia coli, for the enzymatic production of GlcNAc. [Methods] The gene NagZ703 was cloned into the expression vector pET28a to get the recombinant plasmid pET28a-NagZ703. The recombinant NagZ703 was induced for expression in E. coli BL21 (DE3) and purified through His-Trap column. Then the purified NagZ703 was characterized. [Results] The multiple sequence alignments showed that NagZ703 belonged to GH 3 NAGase with 2 domains, and the catalytic active sites were composed of 3 conserved residues (Arg232, His234 and Arg318) at the N-terminal domain. NagZ703 shared only 37% identity with the most studied BsNagZ from B. subtilis. The purified NagZ703 exhibited optimal activity at 60℃ and pH 6.5, the specific activity was 10.79 U/mg, and the K_m and V_max of NagZ703 were 0.276 mmol/L and 0.612 mmol/(mg·min) toward p-nitrophenyl β-N-acetylglucosaminide, respectively. NagZ703 retained more than 80% residual activity after pre-incubation at 50℃ for 30 min, or at pH 6.0-10.5 for 12 h. NagZ703 was a non-metalloenzyme because EDTA could not affect its activity, whereas Hg²⁺ completely inhibited its activity. NagZ703 hydrolyzed colloidal chitin to produce GlcNAc in vitro. [Conclusion] A NAGaseNagZ703 from facultative alkaliphilic Bacillus pseudofirmus was characterized carefully. The ability of NagZ703 to produce GlcNAc from colloidal chitin provided a promising approach for the production of GlcNAc by enzymatic hydrolysis.