Amongst the environmental issues throughout the world, organic synthetic dyes continue to be one of the most important subjects in wastewater remediation. In this paper, the photocatalytic degradation of the dimethylmethane fluorescent dye, Auramine O (AO), was investigated in a heterogeneous aqueous solution with 100 nm anatase TiO2 nanoparticles (NPs) under 365 nm light irradiation. The effect of irradiation time was systematically studied, and photolysis and adsorption of AO on TiO2 NPs were also evaluated using the same experimental conditions. The kinetics of AO photocatalytic degradation were pseudo-first order, according to the Langmuir–Hinshelwood model, with a rate constant of 0.048 ± 0.002 min−1. A maximum photocatalytic efficiency, as high as 96.2 ± 0.9%, was achieved from a colloidal mixture of 20 mL (17.78 μmol L−3) AO solution in the presence of 5 mg of TiO2 NPs. The efficiency of AO photocatalysis decreased nonlinearly with the initial concentration and catalyst dosage. Based on the effect of temperature, the activation energy of AO photocatalytic degradation was estimated to be 4.63 kJ mol−1. The effect of pH, additional scavengers, and H2O2 on the photocatalytic degradation of AO was assessed. No photocatalytic degradation products of AO were observed using UV–visible and Fourier transform infrared spectroscopy, confirming that the final products are volatile small molecules.