Selection of monomer couples, ensuring reactivity ratios close to zero, is an effective strategy to induce spontaneous copolymerization into an alternating sequence. In addition, monomer design and customisation of the solvent–monomer interactions open the way to functional copolymers showing molecular self-assembly relevant to their regular amphipathic structure. In this work, we show that the design of comonomers with adequate reactivities and interactions can be used to direct copolymer self-assembly on a mesoscopic scale. We investigate spontaneous formation of nanoparticles through solvent/non-solvent interactions using the so-called “ouzo effect”. In this way, an ouzo diagram was built to determine the operation window for the self-assembly, in aqueous suspensions, of alternating copolymers consisting of vinyl phenol and maleimide units carrying long alkyl-pendant groups (C12H25 or C18H37). Also, investigations were pursued to account for the influence of the lateral lipophilic pendant units on the size and structure of the nanoaggregates formed during one-shot water addition. Structure characterisation by light scattering techniques (DLS and SLS), small-angle neutron scattering (SANS) and transmission electron microscopy (cryo-TEM and TEM) confirmed the self-assembly of copolymer chains into nanoparticles (size range: 60–300 nm), the size of which is affected by the lipophilicity of the alternating copolymers, solvent–water affinity and the solvent diffusion in water. Altogether, we present here the spontaneous ouzo effect as a simple method to produce stable alternating copolymer nanoparticles in water without the addition of stabilizing agents.