Femtosecond time-resolved optical Kerr effect technique has been used to investigate the third-order nonlinear optical (NLO) properties of a series of conjugated polymers consisting of fluorene and/or tetraphenyldiaminobiphenyl (TPD) units designed to elucidate structure-property relationships for the microscopic second-order hyperpolarizability gamma in polymeric materials. The gamma per repeated unit of the series of polymers has off-resonant values at 830 nm in the range of 2.0 x 10-(33)-2.4 x 10(-31) esu, demonstrating a large modulation of nonlinear optical response by simple structural variations. The gamma values of alternative copolymers containing fluorene and TPD moieties are 2 orders of magnitude higher than the gamma value of the homopolymer polyfluorene, revealing the vital role of the strong electron donor TPD in the NLO enhancement. The fluorene segment was found to result in 4-fold enhancement of gamma in TPD-containing copolymers compared to p-phenylenevinylene segment, indicating that the planar rigid ring of fluorene is an efficient third-order NLO chromophore. No relationship between the magnitude of gamma and the optical band gap was found. The large variation of gamma value with molecular structure of these polymers can be explained by molecular exciton theory other than the band theory.