We report a new method for selective determination of acetaminophen (AP) in physiological condition. A new hybrid film modified electrode was fabricated using inorganic semiconducting nano-TiO2 particles and redox active polymer. Redox polymer, poly(acid yellow 9) (PAY) was electrochemically deposited onto nano-TiO2 coated glassy carbon (GC) electrode. Surface characterizations of modified electrode were investigated by using atomic force microscope and scanning electron microscope. The PAY/nano-TiO2/GC hybrid electrode shows stable redox response in the pH range 1–12 and exhibited excellent electrocatalytic activities towards AP in 0.1 M phosphate buffer solution (pH 7.0). Consequently, a simple and sensitive electroanalytical method was developed for the determination of AP. The oxidation peak current was proportional to the concentration of acetaminophen from 1.2 × 10−5 to 1.20 × 10−4 M and the detection limit was found to be 2.0 × 10−6 M (S/N = 3). Possible interferences were tested and evaluated that it could be possible to selective detection of AP in the presences of dopamine, nicotinamide adenine dinucleotide (NADH), ascorbic acid and uric acid. The proposed method was used to detect acetaminophen in commercial drugs and the obtained results are satisfactory.