Three triphenylamine-fluorene alternating conjugated copolymers, differing only in the aldehyde (PTCHO), monocyano (PTCN), and dicyano (PTDCN) pendant acceptor groups, have been designed and synthesized. The structures and properties of the conjugated copolymers were characterized by H-1 NMR, elemental analysis, gel permeation chromatography (GPC), thermal gravimetric analysis (TGA), UV-visible absorption spectroscopy, photoluminescence (PL) spectroscopy, and cyclic voltammetry (CV). Through manipulating the acceptor attached to the same pi-conjugated backbone, the electronic properties and energy levels of the copolymers were effectively tuned for blue, yellowish-green, and yellowish-orange emissions, resembling those of the primary colors. The effect of substituent on the electronic structure of the copolymers was also studied by molecular simulation. The blue shift in the pi-pi* transition of the backbone was associated with the variation in the structural geometry, whereas the charge transfer, excimer formation, and energy level variation were governed by the electron density distribution. The results suggest a simple and effective approach for tuning the emission in a conjugated polymer through modification of the pendant acceptor groups. In combination with a soluble fullerene (PCBM), PTCN and PTDCN were also used in the fabrication of bulk-heterojunction photovoltaic cells. The structure-property relationships revealed by the present copolymers are useful for the rational design of electroactive polymers for photoelectronic applications.