Miniaturized spectrometers have significantly advanced real-time analytical capabilities in fields such as environmental monitoring, healthcare diagnostics, and industrial quality control by enabling precise on-site spectral analysis. However, achieving high sensitivity and spectral resolution within compact devices remains a significant challenge, particularly when detecting low-concentration analytes or subtle spectral variations critical for chemical and molecular analysis. This study introduces an innovative approach employing guided-mode resonance filters (GMRFs) to address these limitations. Functioning similarly to notch filters, GMRFs selectively block specific spectral bands while allowing others to pass, maximizing energy extraction from incident light and enhancing spectral encoding. Our design incorporates narrow band-stop filters, which are essential for accurate spectrum reconstruction, resulting in improved resolution and sensitivity. Our spectrometer delivers a spectral resolution of 0.8 nm over a range of 370–810 nm. It achieves sensitivity values that are more than ten times greater than those of conventional grating spectrometers during fluorescence spectroscopy of mouse jejunum. This enhanced sensitivity and resolution are particularly beneficial for chemical and biological applications, facilitating the detection of trace analytes in complex matrices. Furthermore, the spectrometer’s compatibility with complementary metal oxide semiconductor (CMOS) technology enables scalable and cost-effective production, fostering broader adoption in chemical analysis, materials science, and biomedical research. This study underscores the transformative potential of the GMRF-based spectrometer as an innovative tool for advancing chemical and interdisciplinary analytical applications.