In this work, we utilise the disulphide bond structure of insulin and a new benzothiazole Raman probe for the detection of human insulin using surface‐enhanced Raman spectroscopy (SERS). The disulphide bond structure of the insulin was reduced to generate free sulfhydryl terminal groups. When reacted with benzothiazole‐functionalised gold nanoparticles, the reduced protein desorbs the Raman probe and causes its Raman signal intensity to quench. Using this approach, insulin was quantified in the concentration range of 1 × 10−14–1 × 10−8 M by SERS quenching. The limit of quantification of insulin by the SERS quenching method was found to be 1 × 10−14 M (0.01 pM or 58 pg/L), which satisfies the requirements for monitoring its blood concentration in patients. Because many proteins and peptides have disulphide bonds in their molecular structures, the new SERS quenching method has a strong potential for the rapid determination of ultralow concentrations of proteins in formulations and biological fluids.