A poly(3-octylthiophene)-block-poly(3-butylthiophene) block copolymer was synthesized in a one-pot block copolymerization reaction, starting from a functional o-tolyl initiator in order to maximize A–B diblock copolymer formation. First, the composition of this block copolymer is extensively studied using gel permeation chromatography (GPC) and ¹H NMR measurements. A complete block copolymer formation is obtained with almost equal block length; B–A–B block copolymer contamination is shown to be very limited. In a second part, the self-assembly was analysed through differential scanning calorimetry (DSC), atomic force microscopy (AFM) and scanning tunnelling microscopy (STM) measurements, focusing on the microphase separation. A direct visualization of the different microphases can be obtained with STM.