A novel fluorescent probe 7-[4′-hydroxy-3′-(5′′-methyl-1H-benzo[d]imidazole-2-yl)styryl]nalidixic acid (HBIN) was synthesized that contains two independent fluorophores and acts as a very sensitive and selective probe for Mg²⁺ and Cu²⁺ ions. Upon addition of Mg²⁺ and Cu²⁺ ions to the solution of HBIN in MeOH–water (9 : 1, v/v), HBIN exhibited a considerable red-shift in its absorption spectrum. Importantly, this novel fluorescent probe exhibited fluorescence enhancement toward Mg²⁺ ions and fluorescence quenching toward Cu²⁺ ions. These changes result from Mg²⁺ and Cu²⁺ ions binding to HBIN at different sites, and undergoing an ICT (intramolecular charge transfer) process and ESIPT (excited-state intramolecular proton transfer) process. Its capability for biological application was also evaluated and the results indicate that HBIN could be successfully employed as a Mg²⁺ and Cu²⁺ ion fluorescent probe in living Hela cells.