Mercury (II) is a potent environmental pollutant with significant health risks. This study presents a spectrophotometric method for the determination of trace amounts of mercury(II) using 3,5-dimethoxy-4-hydroxy benzaldehyde isonicotinoyl hydrazone (DMHBIH) as a complexing agent. The method is based on the formation of a yellowish-brown complex between mercury(II) and DMHBIH in a hydrochloric acid-ammonium hydroxide buffer (pH 6.5). The complex exhibits maximum absorbance at 425 nm, and the method obeys Beer’s law in the concentration range of 0.24–2.88 µg/mL. The molar absorptivity and Sandell’s sensitivity were found to be 2.0 × 104 L mol-1 cm-1 and 0.00362 µg/cm-2, respectively. The method was successfully applied to determine mercury (II) in liver samples, demonstrating its applicability in real-world scenarios.