A resonance Rayleigh scattering (RRS) technique was utilized as a tool for isoelectric point monitoring and iron(III) cation determination. The spectral properties of some amphoteric molecules (proteins and a DNA sequence) were investigated using the RRS technique. When the pH values were kept at around their isoelectric points, especially high RRS signals could be obtained, which were much stronger than those at other pH values. By using the C30 DNA sequence as a probe, the iron(III) cation can be detected rapidly. After iron(III) was added to a C30 solution, a significantly decreased RRS signal was obtained. The sensing process can be finished within 10 min with a detection limit of 0.9 μM. Thus, a sensitive, selective, and label-free method was successfully developed for iron(III) detection.