AbstractA new 1,10‐phenanthroline (Phen) derivative, 3,8‐bis(5‐methylthiophen‐2‐yl)‐1,10‐phenanthroline (PHT1), has been synthesized and fully characterized including by single‐crystal X‐ray structure analysis. The electron‐donating effects of the two 5‐methylthiophene groups lead to distinctive fluorescence emission enhancement on complexation with Zn2+ compared with non‐emissive Phen. However, PHT1 shows stronger Zn2+‐sensing ability than 3,8‐bis(thiophen‐2‐yl)‐1,10‐phenanthroline (PHT0) or 3,8‐bis(3‐methylthiophen‐2‐yl)‐1,10‐phenanthroline (PHT2) and longer‐wavelength fluorescence emission (λem = 461 nm), which is vital for studies of living systems, mainly because of the lower steric hindrance and the electron‐donating effects of the 5‐methyl groups on the thiophene rings. More interestingly, PHT1 displays high selectivity towards Zn2+ over a broad range of pH and has an extremely low detection limit (5 ppb). An approximately 51‐fold Zn2+‐selective CHEF response has been attributed to the formation of a 1:2 metal–ligand complex, and the association equilibrium constant (Kass0) of [Zn(PHT1)2](ClO4)2 was determined to be 3.4 × 1012. Anionic effects and variations in the 1H NMR spectra before and after zinc(II) ion complextion were observed. Moreover, the ability of probe PHT1 to sense Zn2+ within living HL‐60, yeast, and HepG‐2 cells has been explored, and the Zn2+‐probing process in living cells was found to be reversible with a zinc chelator solution of N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN).