The correlation between the molecular design of a conjugated polyelectrolyte (CPE) and its aggregated structure and the emissive properties in water is systematically investigated by means of UV–vis spectrometry, fluorescence spectroscopy, and scanning/transmission electron microscopy. Five different and rationally designed CPEs having carboxylic acid side chains are synthesized. All five conjugated polyelectrolytes are seemingly completely soluble in water in visual observation. However, their quantum yields are dramatically different, changing from 0.45 to 51.4%. Morphological analysis by electron microscopy combined with fluorescence spectrophotometry reveals that the CPEs form self-assembled aggregates at the nanoscale depending on the nature of their side chains. The feature of the self-assembled aggregates directly determines the emissive property of the CPEs. The nature and the length of the spacer between the carboxylic acid group and the CPE backbone have a strong influence on the quantum yield of the CPEs. Our study demonstrates that bulky and hydrophilic side chains and spacers are required to achieve complete water-solubility and high quantum yield of CPEs in water, providing an important molecular design principle to develop functional CPEs.