New rod-coil copolymers based on poly(p-phenylene benzobisoxazole) (PBO) have been synthesized and characterized to explore a new ordered molecular structure at the supramolecular level for regulating photophysical properties and obtaining new blue light-emitting materials. The microstructure and physical properties of such rod-coil copolymers, poly(1,4-phenylenebenzobisoxazole-co-octa-methylenebenzobisoxazole) (PBO-PBOC8), were determined by FTIR, H-1 NMR, viscometry, WAXD, and TGA. The chain flexibility and photophysical properties of copolymers can be efficiently modulated by the copolymer composition while the structure remains ordered, which is quite different from their analogue: the amorphous rod-coil copolymers poly(1,4-phenylenebenzobisthiazole-co-decamethyleneben-zobisthiazole) (PBZT-PBTC10) reported by Jenekhe et al. [J. Am. Chem. Soc. 1995, 117, 7389]. Photophysical properties of these polymers both in methanesulfonic acid (MSA) and in the solid state were investigated in detail using UV absorption, photoluminescence (PL) spectroscopy, and time-resolved PL spectroscopy to reveal the relation between the morphology and photophysical properties. The PL peak of the rod-coil copolymers was greatly blue-shifted from that of the pure conjugated polymer PBO, particularly at rod molar fractions of less than 0.5. Furthermore, electronic paramagnetic resonance (EPR) studies of the paramagnetic defect in the copolymers were performed to explore the effect of structural change on the EPR signal.