Objective To optimize the synthesis approach of 6-methyl-2-pyridyl N-acetyl-1-thio-β-D-glucosaminide (MPT-NAG) as a novel substrate for N-acetyl-β-D-glucosaminidase (NAG), and explore its value in diagnosis of renal tubular lesions. Methods The substrate was prepared by the use of 1-chloro-1-deoxy-tetraacetyl-D-glucosamine and 2-mercato-6-methylpyridine in glycoside reaction, and urinary NAG activities of samples from patients with diabetic nephropathy, patients with nephrotic syndrome, patients with renal transplantation and normal controls were detected by automated analytical device with MPT-NAG as substrate. Results The structure of the substrate of MPT-NAG was identified by elemental analysis and 1HNMR, and the purity was 99.8% determined by high performance liquid chromatography (HPLC). It was revealed that the urinary NAG activities in patients with diabetic nephropathy, nephrotic syndrome and renal transplantation were significantly higher than those in the normal group (P<0.01). Conclusion The synthesized novel substrate of MPT-NAG obtained by optimized approach with low cost and high production can be used in the diagnosis of renal tubular lesions.