An electrochemiluminescent (ECL) biosensor was constructed for selective assay of alanine aminotransferase (ALT) based on the enzymatically catalyzed oxidation of pyruvate by pyruvate oxidase (PYOD). The composite of potassium ferricyanide and carbon nanotube was adopted to pre-functionalize the basal platinum electrode while the potassium ferricyanide acted as the activator of PYOD. The ALT catalyzed the reaction of L-alanine and-ketoglutarate to produce pyruvate which could be further enzymatically oxidized by PYOD to yield H2O2 to intensify the ECL of luminol. The biosensor showed rapid response for real-time measurement of ALT in the linear concentration range from 0.00475 to 350 U/L (r = 0.993) with a relatively standard deviation of 2.5% (CALT = 47.5 U/L,n = 6). The biosensor was applied to assay the ALT in rat serum with average recovery of 90.5%. An electrochemiluminescent (ECL) biosensor was constructed for selective assay of alanine aminotransferase (ALT) based on the enzymatically catalyzed oxidation of pyruvate by pyruvate oxidase (PYOD). The composite of potassium ferricyanide and carbon nanotube was adopted to pre-functionalize the basal platinum electrode While the potassium ferricyanide acted as the activator of PYOD. The ALT catalyzed the reaction of L-alanine and-ketoglutarate to produce pyruvate which could be further enzymatically oxidized by PYOD to yield H2O2 to intensify the ECL of luminol. The biosensor showed rapid response for Real-time measurement of ALT in the linear concentration range from 0.00475 to 350 U / L (r = 0.993) with a relatively standard deviation of 2.5% (CALT = 47.5 U / L, n = 6). The biosensor was applied to assay the ALT in rat serum with average recovery of 90.5%.