Abstract: The measurement of serum glucose represents a fundamental experiment in clinical biochemistry laboratory courses. However, current methodologies present several drawbacks, including the utilization of highly toxic chromogenic substrates, limited anti-interference capacity, and time-consuming instrumental analysis. This enhanced experimental protocol maintains the fundamental principle of enzyme cascade catalysis while implementing improvements in three critical dimensions: 1) substitution of the highly toxic 4-aminoantipyrine chromogen with the less hazardous 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), substantially improving operational safety; 2) development of a stable and sensitive nanoprobe through biomimetic enzyme immobilization technology, effectively enhancing both interference resistance and detection accuracy while elevating the experiment’s innovative and advanced characteristics; 3) replacement of traditional UV-Vis spectrophotometry with high-throughput microplate reader technology, significantly reducing analysis time and sample volume requirements. Teaching evaluations demonstrate that the synthesized nanoprobe features straightforward preparation, cost-effectiveness, excellent reproducibility, and superior detection accuracy. This optimized experiment successfully integrates teaching safety, advanced concepts, and cutting-edge technology. Its modular design enables completion within 7–10 instructional hours, offering a replicable model for reforming clinical biochemistry laboratory curricula.

Key words: Serum glucose, Enzyme immobilization, Nanoprobe, Colorimetric analysis, Experimental improvement