Carboxylated poly(styrene-b-ethylene-co-butylene-b-styrene) (CSEBS) with different carboxylation extents [-COOH] were prepared. The [-COOH] dependence of the self-assembly of CSEBS in water was investigated by a combination of static and dynamic laser light scattering. The addition of a dilute CSEBS tetrahydrofuran solution dropwise into water leads to a microphase inversion, resulting in surfactant-free polymeric nanoparticles stable in water. As [-COOH] increases, the particle size first decreases and then slightly increases; namely, there exists a minimum in particle size when [-COOH] approximate to 21 mol %. On the other hand, the decrease of the average particle density [rho] and the increase of the ratio of the average radius of gyration to the average hydrodynamic radius ([R-g]/[R-h]) as [-COOH] increases reveal that the CSEBS nanoparticle gradually changes from a uniform and compact globule to a hyperbranched and loose cluster. The cluster can be broken by a simple dilution of the dispersion, indicating that there exists a delicate balance between hydrophobic and hydrophilic interactions in the self-assembly.