Chronic otitis media is a recurrent middle ear infection that poses a common health risk globally, particularly in the developing countries. Association between heavy metals, specifically copper and lead and otitis media has been underexplored. Herein, a spectrofluorometric method was developed to determine copper and lead in human plasma samples investigating their association with chronic otitis media. The method utilizes feasible interaction of 6,7-dihydroxy-4-methylcoumarin, with copper and lead to yield different fluorescent compounds that provides measurable approaches for their levels in the plasma matrix. Copper is quantitatively analyzed based on its quenching ability for the fluorescence of 6,7-dihydroxy-4-methylcoumarin exhibiting concentration-dependent behavior. On the other hand, lead forms a fluorescent complex with 6,7-dihydroxy-4-methylcoumarin that has remarked fluorescence enhancement and bathochromic shift, enabling sensitive lead quantification. The method was briefly validated and introduced accepted linear relationship over a concentration range of 100–3000 ng/mL for copper and 200–4000 ng/mL for lead. The method was used to determine the levels of copper and lead in the human plasma samples of healthy volunteers and patients with chronic otitis media. The results revealed a decrease in copper levels and an increase in lead levels in otitis media patients compared to healthy controls. These findings suggest a potential association between copper and lead levels and chronic otitis media, warranting further investigation into their role in the condition.