Hydrophobically modified pectin derivatives were prepared by immobilization of long alkyl chains (C-12-C-18) at various substitution ratios, using two different synthetic pathways, one affording covalent fixation and the other one leading to a mere ionic association. These derivatives display an associative tendency in semidilute as well as in dilute aqueous solutions. This phenomenon, which stems from both intra- and intermolecular interactions between hydrophobic groups, results in the formation of hydrophobic microdomains. The latter can be characterized, especially in the dilute regime, thanks to fluorescence spectroscopy. Fluorescent molecular rotors, as well as pyrene, a classical fluorescence probe of widespread use, witness the variations of the medium polarity. In addition, they can also provide further information, particularly about the local cohesion of the microenvironment of the probe, without performing any complementary experiment, for example, the addition of quenchers together with the fluorescent probe in the polymer solutions. CAC values derived from polarity changes (CAC(polarity)), using the molecular rotor as well as pyrene as the fluorescence probes, are significantly different from those determined from the cohesion of the microenvironment (CAC(cohesion)), accessible only with the molecular rotor. This latter type of fluorophore may therefore enable us to determine more accurately the actual critical aggregation concentration.