In this study, we introduce a high-performance front-light structure integrated with optical polarization control to improve low-contrast fluorescent imaging in lensless devices. Lensless fluorescence imaging is an advanced technology with the potential to enhance the performance of biological and neurological imaging. However, there are still challenges to achieving high image quality, particularly in low-contrast fluorescent signals due to light scattering and absorption. The performance of a low-fluorescence stripe diffractor, fabricated using Norland Optical Adhesive 63 (NOA 63), was evaluated in comparison to polydimethylsiloxane (PDMS) material as a light guide. The diffractor is designed to operate with a hybrid emission filter on the image sensor. As a result, by coupling a 450 nm laser with either P- or S-polarization, it was shown that NOA 63 is notable as a diffractor material. Notably, S-polarized light showed a great excitation intensity and an enhanced contrast in fluorescent bead imaging compared with P-polarized light. Furthermore, the system was validated for biological applications by successfully detecting fluorescent bead emission in agarose-embedded samples of various thicknesses, simulating brain tissue. These results emphasize the system’s ability to capture low-intensity fluorescence signals from deep layers of biological tissue.