According to the refractive index change of glass, sliver and polystyrene for different incident wavelengths, the sensitivity of surface plasmon resonance (SPR) sensor for different wavelengths were analyzed. The experimental data are used to fit the refractive index change of glass, sliver and polystyrene with wavelength, and the SPR sensor sensitivity under different wavelengths was achieved by application of the fit result in theoretical calculation. The optimized wavelengths under different sensitivity requirement of SPR sensor were proposed. To prove the theoretical result, the light sources with wavelengths of 568 nm and 632.8 nm were applied to SPR sensor. The two light sources were used to achieve sample surface morphology, and the result was found that the detail of sample was better presented by the light source with wavelength of 632.8 nm than that with 568 nm wavelength.