We have successfully developed a bis-schiff base fluorescent probe L, linking hemicyanine aldehyde and salicylaldehyde through hydrazine hydrate, which continuously identify Fe3+ and F− in MeCN-Tris (v/v, 2:1, 10 mM, pH 7.3) solution. The probe L showed a highly selective and sensitive fluorescence "turn-on" for Fe3+ over other metal ions accompanying with the solution color change from yellow to red. Furthermore, the resulting L-Fe3+ complex detect only F− among the common various anions. The bonding mechanisms were detected by fluorescence and absorption titrations, 1H NMR titrations, HRMS and theoretical calculations. In addition, the probe has been used for liquid colorimetry and test paper colorimetric identification of Fe3+. Moreover, the probe L was used for sequentially off-on-off fluorescence imaging of Fe3+ and F− in HeLa cells. In this study, we have successfully developed a bis-schiff base fluorescent probe L, linking hemicyanine aldehyde and salicylaldehyde through hydrazine hydrate, which continuously identify Fe3+ and F− in MeCN-Tris (v/v, 2:1, 10 mM, pH 7.3) solution. The probe L showed a highly selective and sensitive fluorescence "turn-on" for Fe3+ over other metal ions accompanying with the solution color change from yellow to red. Furthermore, the resulting L-Fe3+ complex detect only F− among the common various anions. The bonding mechanisms were detected by fluorescence and absorption titrations, 1H NMR titrations, HRMS and theoretical calculations. In addition, the probe has been used for liquid colorimetry and test paper colorimetric identification of Fe3+. Moreover, the probe L was used for sequentially off-on-off fluorescence imaging of Fe3+ and F− in HeLa cells. [Display omitted] • A new hemicyanidine-based colorimetric-fluorescent probe L was synthesized. • The probe L can detect Fe3+ and F− ions in aqueous solutions. • The binding modes of probe L with Fe3+ have been well demonstrated by MS, 1H NMR and DFT calculation. • The probe L had the potential application value for monitoring Fe3+ and F− ions in environment and vivo. [ABSTRACT FROM AUTHOR]