In our work, one-step electro-deposition method was adopted to produce polyaniline (PANI) and functional multiwalled carbon nanotubes (f-MWCNTs) films on glass carbon electrodes, and the modified electrodes were applied as an electrochemical sensor for determination of 10-hydroxycamptothecine (10-HCPT). The f-MWCNTs were handled by ultrasound processing in concentrated oxidizing acid solution, which can obtain a wonderful dissolution in water and attach new functional groups, such as –COOH and –OH. Then, aniline monomer could polymerize on the surface easily. The surface characterization was investigated using various techniques including scanning electron microscope, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction, and electro-catalytic properties were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Under optimal conditions, the resulting of PANI/f-MWCNTs sensor showed a wide linear range (3 × 10−9 to 7 × 10−7 mol L−1) and a low detection limit (1 10−9 mol L−1), which is attributing to its large special surface area and good conductivity. Moreover, the modified electrodes are convenient to fabricate, which can be used to detect 10-HCPT in urine samples successfully.