Bathophenanthroline (BPhen) is a versatile bidentate ligand for transition metals, also frequently used as a universal colorimetric probe for Fe(II). The effect of pH on the fluorescence intensity of BPhen and quenching constant of Fe(II) were studied at different pH values using working solutions of 50% ethanol buffered with acetate buffer. Fluorescence intensity of a 3.0 μM Bphen solution in 50% ethanol buffered at pH 6.6 with acetate buffer was selectively quenched by Fe(II) at 25 °C. The LOD of 19 nM at 3.3 σ and the linear range of 63 nM – 224 nM with R2 = 0.9919 revealed that this method is more sensitive than the colorimetric method (detection range =1.8 μM - 18 μM). Quenching of the BPhen-Fe(II) complex is temperature-dependent, which may be due to the increased stability of the formed complex with temperature, supporting a static quenching mechanism. Interference from foreign ions on the fluorescence of the BPhen-iron(II) complex was also studied. A tolerance limit exceeding 5% was observed and recorded. The interference from cations, Ni(II), Co(II), and Cu(II), was relatively higher, while the most common anions showed only a little to no interference. In response time analysis, the fluorescence intensity, which remains constant after 10 minutes, reveals that the system approached equilibrium. This method would be quicker and more accurate than a colorimetric method due to the absence of a solvent extraction step.