The chemiluminescence assay of urinary N-acetyl-β-D-glucosaminidase using o-aminophthalylhydrazido-N-acetyl-β-D-glucosaminide as the substrate was examined. Under optimized conditions, a linear chemiluminescence response to the standard N-acetyl-β-D-glucosaminidase (NAGase) of different concentrations was observed with a correlation coefficient of 0.999 over the range of 0.3 to 20 I.U./l. The present method was compared with the prevalent method using p-nitrophenyl-N-acetyl-β-D-glucosaminide in the assay of 20 urine samples from healthy subjects, and the correlation coefficient of 0.891 was obtaiend.