a combined carbonization-selenization strategy. Its application as a nanozyme for the highly sensitive colorimetric detection of L-cysteine (L-Cys) was systematically investigated. The synergistic effect between FeSe nanoparticles and nitrogen-doped porous carbon (NC) significantly enhances the catalytic performance toward L-Cys. FeSe nanoparticles serve as a pivotal source of abundant active sites to facilitate redox reactions, while the NC matrix, with its high specific surface area and uniformly distributed nitrogen-doped sites, effectively improves electron transfer efficiency. Under optimized experimental conditions, the FeSe-NC-900-based colorimetric sensor exhibits a linear detection range for L-Cys from 1 to 50 µM, with a low limit of detection (LOD) of 0.5613 µM (S/N = 3). Moreover, the sensor demonstrates excellent selectivity against common interfering substances, which is attributable to the specific interaction between FeSe active sites and L-Cys. To validate its practical applicability, the FeSe-NC-900 sensor was employed for L-Cys detection in sheep serum samples, yielding recovery rates ranging from 89.6% to 99.8%. These results confirm its reliability and potential for use in complex biological matrices.