Nucleation and growth mechanism of electropolymerization of methylene blue (MB) in a basic medium and the effect of preparation potential on poly(MB) film structure were investigated by using cyclic voltammetry, potentiostatic current-time transient, scanning tunneling microscopy (STM), atomic force microscopy (AFM), and UV-vis. absorption spectroscopy techniques. Electropolymerization of MB has been achieved by potentiodynamic (cyclic voltammetry) and potentiostatic (constant potential) techniques. The potentiostatic current-time transients fitted with a theoretical model and morphological studies indicate that nucleation and growth mechanism of poly(MB) starts with a progressive layer-by-layer nucleation and growth besides random adsorption. Nucleation and growth of poly(MB) follows a process between progressive layer-by-layer and 3-D instantaneous mechanism resulting in highly-oriented poly(MB) nanofibers with increasing poly(MB) film thickness. Cyclic voltammetry and morphological studies exhibit that poly(MB) film structure changes depending on the preparation potential. Poly(MB) films prepared at the potential values of 900 and 950 mV show a well-ordered, smooth surface but at the potential values higher than 1000 mV, rough polymer surface arises as overoxidation takes place. UV-vis. absorption spectra of poly(MB) film and MB monomer show three peaks. The peak at 410 nm for poly(MB) shows 100 nm blue shift when compared to the MB monomer and is attributed to poly(MB) formation on the electrode.