Abstract The fabrication of discrete chiral Au nanorods (c‐Au NRs) used as new emerging chiral plasmonic nanozyme for colorimetric detection of glucose is presented. It is found that the colorimetric detection of glucose based on the c‐Au NRs is dependent on chiral geometries and polarized light. In comparison to Lc‐Au NRs, Dc‐Au NRs possess a lower linear concentration range of glucose detection and limit of detection (LOD) due to their higher affinity capacity. More importantly, polarized light (LP: linearly polarized light; LCP: left‐handed circularly polarized light; RCP: right‐handed circularly polarized light) can enantioselectively and markedly improve the LOD of glucose due to circularly polarized hot electrons and electromagnetic field. For instance, the LOD is lower under the RCP irradiation for Dc‐Au NRs in comparison to that under LCP irradiation. Similarly, the LOD is higher under the RCP irradiation for Lc‐Au NRs than that under LCP irradiation. Our work not only provides a novel strategy for the detection of glucose levels, but also reveals the chiral interaction between chiral matters and chemical substrates, which will be helpful in the field of chiral recognition, chiral catalysis, and chiral drugs.