Hydrogen sulfide (H2S) plays dual-faced roles in physiological regulation and food quality, demanding detection tools with high sensitivity, specificity, and self-validating capability. Herein, a dual-mode biosensor integrating fluorescence (FL) and surface-enhanced Raman scattering (SERS) is developed for ultrasensitive detection and mitochondrial-targeted imaging of H2S. The biosensor is constructed based on an azide-functionalized metal-organic framework (UiO-66-N3) coupled with gold nanoparticles (AuNPs), where the electron-withdrawing -N3 group serves simultaneously as an FL quencher and a SERS reporter. Upon H2S exposure, the selective reduction of -N3 to -NH2 triggers an FL turn-on response and a concomitant SERS signal attenuation. This dual-response mechanism allows for quantitative H2S detection across a wide dynamic range (0.01-500 nM), with detection limits reaching as low as the pM level. Further functionalization with a mucin1 aptamer and a mitochondrial localization sequence empowers tumor-specific mitochondrial targeting and high-fidelity imaging of endogenous H2S. The biosensor demonstrates robust applicability in complex beer and wine samples and enables real-time tracking of mitochondrial H2S fluctuations in living cells. This work establishes a versatile and self-validating sensing platform that bridges materials design with practical bioanalysis, highlighting its broad potential in food safety monitoring and biomedical diagnostics.