Herein, we report a new pyridine-biquinoline-derivative fluorophore L for effectively sensing pyrophosphate (PPi) and monohydrogen sulfide (HS–) in aqueous buffer and in living cells. L could selectively coordinate with metal ions (Mn+) in Groups IB and IIB to form L-Mn+ complexes with 1:1 stoichiometry, resulting in fluorescence quenching via photoinduced electron transfer (PET) mechanism. L-Zn2+ complex was applied to competitively coordinate with PPi to form a new “ate”-type complex, turning on the fluorescence by a 21-fold-increase. The limit of detection (LOD) of this assay for PPi detection in aqueous buffer is 0.85 μM. L-Cu2+ complex was applied for highly selective detection of HS– with an excellent sensitivity by 25-fold decomplexation-induced fluorescence increase. LOD of L-Cu2+ complex for HS– detection in aqueous buffer is 2.24 μM. With the in vitro data obtained, we successfully applied these two complexes for sequential imaging Zn2+ and PPi, Cu2+ and HS– in living cells, respectively. Since PPi and HS– occur in vascular calcification in positive correlation, our multifunctional probe L might help doctors to more precisely diagnose this disease in vivo in the future. For example, we could use radioactive tracer L-64Cu for qualitative and quantitative positron emission tomography/computed tomography (PET/CT) imaging of HS– in vivo.