A direct, extraction-free spectrophotometric method was developed for the determination of benzalkonium bromide (BAK). The method is based on the formation of mixed dye–surfactant aggregates between alizarin green (AG) and BAK in alkaline medium by measuring the decrease in absorbance of AG at 460 and 700 nm. Beer’s law was obeyed in the concentration range 3–40 μg mL−1 with good precision and accuracy. The limits of detection were 0.4 μg mL−1 at 460 nm and 0.3 μg mL−1 at 700 nm, which reduced to 0.2 μg mL−1 by combining the absorbance at the two wavelengths. The proposed method was successfully applied to the determination of BAK in disinfectant solution and eye drops. The analytical results of the real samples were in good agreement with those of an HPLC method.