The effect of electric field application on the optical properties of gold (Au) thin film for spectroscopic ellipsometry-based surface plasmon resonance (SPR) biosensor applications has been successfully studied. The optical properties, such as the complex refractive index, complex dielectric constant, real optical conductivity, and absorption coefficient, were determined by fitting data from the ψ and Δ functions obtained through measurements using a homemade rotating analyzer ellipsometer. The applied electric fields of 0.8 V/cm, 1.6 V/cm, 2.4 V/cm, and 3.2 V/cm were induced by applying voltages of 1 V, 2 V, 3 V, and 4 V for ellipsometer tests, and 1.6 V/cm to 3.2 V/cm for the SPR test. The Au thin film was deposited onto a glass slide using an evaporator for ellipsometer testing and a BK7 half-cylinder prism was employed for SPR testing. The experimental results show that the real part of the refractive index (n) increases in the spectral range from 1.5 eV to 2.2 eV and from 3 eV to 5 eV and tends to be constant in the range from 2.3 eV to 2.8 eV. Conversely, the imaginary part of the refractive index (k) decreases from 2.8 eV to 3.6 eV and tends to remain consistent from 3.9 eV to 5.0 eV. The dielectric constant (ε1) consistently shows a significant increase within the spectral range of 3 eV–5.0 eV, with a tendency to decrease between 2.1 eV and 2.4 eV. Meanwhile, the imaginary part (ε2) demonstrates an increase in the spectral range of 1.5 eV–2.4 eV and 3.3 eV–5.0 eV while maintaining relatively stable values in the energy spectral range of 2.6 eV–3.0 eV. Moving to the real part of optical conductivity (σ1), it increases within the spectral range from 1.5 eV to 2.4 eV and from 3.5 eV to 5 eV, with relatively constant values between 2.6 eV and 3.1 eV. The absorption coefficient (α) exhibits a decrease in the spectrum between 2.8 eV and 3.6 eV, but the values remain stable within the range of 3.9 eV–5.0 eV. The α slightly decreases in the energy range of 1.8 eV–2.6 eV. The outcomes obtained from these experiments are directly linked to the SPR results, revealing a noticeable shift in the SPR angle (θSPR) of 45.51°, 45.61°, and 45.73° and an increase in the minimum reflectance (Rmin) of 0.05, 0.09, and 0.13 for electric field strengths of 0 V/cm, 1.6 V/cm, and 3.2 V/cm, respectively. These results imply that the application of an external electric field to the surface of the Au thin film disrupts the matching condition between the evanescent wave vector and the surface plasmon wave vector, thereby altering the reflected light under resonance conditions indicated by an increase in the minimum reflectance and SPR angle. This development holds significant promise to further enhance the sensitivity of electro-optic modulation-based biosensors.