A benzimidazole–based chemosensor (DFB) for Cu2+ and Zn2+ was developed, and its sequential anions (CN− and P2O74-) and DNA sensing capability and its live-cell imaging, as well as its PPi detection in PCR-amplified DNA products, has been demonstrated. Display omitted •A benzimidazole-based chemosensor (DFB) shows “off-on” responses upon differential detection of Cu2+ and Zn2+.•The DFB derived Cu2+ & Zn2+ complexes act as efficient sensors toward CN−, PPi and DNA in an aqueous medium.•DFB acts as a sequential fluorescent probe to detect Cu2+, Zn2+ and PPi in living cancer cells.•DFB-Zn2+ can detect the PPi in the PCR amplified DNA products. Differential chemosensors have emerged as next-generation systems due to their simplicity and favourable responsive properties to produce different signals upon selective binding of various analytes simultaneously. Nevertheless, given their inadequate fluorescence response and laborious synthetic procedures, only a few differential chemosensors have been developed so far. In this work, we have employed a single pot synthesis strategy to establish a new benzimidazole-based Schiff base type fluorogenic chemosensor (DFB) which differentially detects Cu2+ (detection limit (LOD) = 24.4 ± 0.5 nM) and Zn2+ (LOD = 2.18 ± 0.1 nM) through fluorescence “off-on” manner over the library of other metal cations in an aqueous medium. The DFB-derived ‘in situ’ complexes DFB-Cu2+ and DFB-Zn2+ showed fluorescence revival “on-off” responses toward cyanide (CN−) and bio-relevant pyrophosphate (P2O74--PPi) ions with a significantly low LOD of 9.43 ± 0.2 and 2.9 ± 0.1 nM, respectively, in water. We have demonstrated the phosphate group-specific binding capability of DFB-Zn2+, by testing it with both ssDNA and dsDNA samples which displayed fluorescence “turn-off” response (LOD ∼10-7 M), similar to the PPi binding in an aqueous medium, indicating that it interacts explicitly with the phosphate backbone of DNA. We have also harnessed the DFB as a sequential fluorescent probe to detect Cu2+, Zn2+, CN− and P2O74- ions in human cervical (HeLa) and breast (MCF-7 and MDA-MB-231 (aggressive and invasive)) cancer cell lines. Moreover, we have explored the PPi recognition capability of DFB-Zn2+ in the polymerase-chain-reaction (PCR) products where PPi is one of the primary by-products during amplification of DNA.