In this study, zinc ions were employed as the central metal, while 4,4',4''-nitrilotribenzoic acid (H3NTB) served as the fluorescent organic ligand. Through a single-step solvothermal approach, a novel Zn-based metal-organic framework (Zn-MOF) fluorescent material was successfully synthesized. The Zn-MOFs exhibit a cubic morphology, outstanding fluorescence properties, excellent water dispersibility, and robust resistance to high temperature and strong alkaline conditions. Additionally, MnO2 nanosheets (NSs) were effectively exfoliated from MnO2 particles using ultrasonic treatment. These MnO2 NSs possess a broad UV-Vis absorption band that overlaps with the fluorescence spectra of Zn-MOFs. Leveraging the inner filter effect (IFE) between MnO2 and Zn-MOFs, a Zn-MOF-based fluorescence bioassay technique was developed for the detection of biothiols. The results demonstrate that this Zn-MOF-based fluorescence detection platform exhibits a remarkable sensitivity towards biothiols, achieving a detection limit of 0.067 μM, surpassing that of other reported MnO2-based detection methods. Furthermore, this detection platform has been successfully applied to the quantitation of glutathione (GSH) in human serum, highlighting its potential for highly sensitive and specific detection of biothiols.