The performance of a novel ion-selective membrane electrode for the determination of mercury(II) based on a tribromomercurate–rhodamine B (TBM–RB) ion-pair complex has been described. The developed electrode showed a linear, reproducible, and stable potentiometric response with an anionic super-Nernstian slope of 69.5 ± 0.5 mV per decade over a wide range of concentration, 10−5–10−2 M , with a detection limit of 2.4 × 10−6 M of Hg2+. The membrane showed a fast response time of 10–20 s and in a pH range of 1.5–7.5. The proposed sensor exhibited excellent selectivity for the tribromomercurate anion over anions: Br−, Cl−, , , acetate, oxalate, citrate, sulphate, phosphate, selenite, and nitrate. Also, there is a negligible interference from different cations such as, Ca2+, Sr2+, Cu2+, Ni2+, Co2+, Fe2+, Mn2+, Pb2+, Al3+, and Zn2+. The prepared sensor was used successfully as an indicator electrode for potentiometric titrations of mercury(II) with rhodamine B and thiosulphate. The analysis of mercury(II) ions in wastewater and microbial media with average recovery percentages of 100.6 ± 1.8% and 101.3 ± 1.7%, respectively, using the developed sensor was in good agreement with the data obtained using the standard dithiazone spectrophotometric method.