Dumbbell-like nanoparticles, previously known for their excellent light absorption and dispersion, have diverse applications, especially in SERS. Herein, we have explored a fast and novel controlled synthesis approach to achieve a high formation of AuNDBs. Precise control of precursor amounts and seed addition order halted seed growth at the AuNDB formation stage, preventing further elongation into rod-like structures. The AuNDBs were functionalized with 4-mercaptobenzoic acid (4-MBA) to create SERS nanosubstrates and deposited on the glass slide through 3-mercaptopropyl (dimethoxy)methylsilane by the spin-coating technique. The detection of d-glucose was achieved by measuring the signal decrease of the 4-MBA Raman probe molecules on the nanosubstrates at different trace concentrations of the analyte. These novel SERS substrates have the potential to provide an enhancement factor (EF) of 2.41 × 107 and a limit of detection (LOD) as low as 1.17 nmol/L. The electromagnetic field distribution of AuNDBs with many arrangements surrounded by air and d-glucose solutions was also determined by using the finite-difference time-domain technique (FDTD). The FDTD results confirmed that d-glucose on the SERS substrates caused the local electromagnetic field to degrade, resulting in the degree of the Raman signal. Our findings suggest that dumbbell-shaped SERS substrates can effectively detect very low levels of glucose, making them ideal for designing novel anisotropic nanoparticle-functionalized surfaces with ease.