Mercury detection in water has attracted a lot of research interest due to its highly toxic nature and adverse environmental impact. In particular, the recent discovery of specific binding of HgII to thymine-rich (T-rich) DNA resulting in T-HgII-T base pairs has led to the development of a number of sensors with different signaling mechanisms. However, the majority of such sensors were non-immobilized. Immobilization, on the other hand, allows active mercury adsorption, signal amplification, and sensor regeneration. In this work, we immobilized a thymine-rich DNA on a magnetic microparticle (MMP) surface through biotin–streptavidin interactions. In the presence of HgII, the DNA changes from a random coil structure into a hairpin, upon which SYBR Green I binds to emit green fluorescence. Detection was carried out by using flow cytometry where the fluorescence intensity increased ≈9-fold in the presence of mercury and the binding of mercury reached equilibrium in less than 2 min. The sensor showed a unique sample-volume-dependent fluorescence signal change where a higher fluorescence was obtained with a larger sample volume, suggesting that the particles can actively adsorb HgII. Detection limits of 5 nM (1 ppb) and 14 nM (2.8 ppb) were achieved in pure buffer and in mercury-spiked Lake Ontario water samples, respectively.