${eta}$-N-Acetyl-D-glucosaminidase B was highly purified with the following sequence of steps; DEAE-cellulose, CM-cellulose, and Sephadex G-200 gel filtration chromatograpies. The specific activity of the purified ${eta}$ -N-acetyl-D-glucosaminidase B was 2.2 units/mg protein with 12.9 % yield and 196.2 fold purity. The purified ${eta}$-N-acetyl-D-glucosaminidase B showed single band on polyacrylamide gel electrophoresis. The final preparation of ${eta}$ -N-acetyl-D-glucosaminidase B was completely free friom arylsulfatase and ${eta}$-glucuronidase. ${eta}$ -N-Acetyl-D-glucosaminidase B had pH optimum of 4.5 in 0.5 M sodium citrate buffer. The molecular weight of ${eta}$-N-acetyl-D-glucosaminidase B was 133,000 by Sephadex G-200 gel filtration. The Km value of ${eta}$-N-acetyl-D-glucosaminidase B using p-nitrophenyl-N-acetyl-${eta}$-D-glucosaminide as substrate was 1.0 mM and $V_{max}$ was 0.014 ${mu}$ mole/min. ${eta}$-N-Acetyl-D-glucosaminidase B was stable at $55^{circ}C$ for 70 minutes. The crude ${eta}$ -N-acetyl-D-glucosamiinidase in 70 % ammonium sulfate retained 93 % activity after 7 months storage at -$55^{circ}C$. Bovine serum albumin, sodium chloride, and phosphate activated ${eta}$ -N-Acetyl-D-glucosaminidase B. N-Acetyl-D-glucosamine, ${alpha}$-methyl-D-mannoside, and acetate inhibited ${eta}$ -N-acetyl-D-glucosaminidase B.