In this study, a three-dimensional (3D) polyacrylamide microstructure containing gold nanorods (AuNRs) was fabricated successfully by utilizing femtosecond laser-based two-photon polymerization (TPP) with a Rose Bengal (RB) photoinitiator, and can provide a great diversity of optical properties. To maintain AuNRs in the 3D polymer microstructures, the fabrication laser power can be significantly reduced to 1.0 mW by tuning the laser wavelength for the two-photon absorption of RB to improve TPP efficiency, but not for the longitudinal plasmon resonance of AuNRs to photothermally damage AuNRs. After the TPP processing, a higher laser power, greater than the threshold of the AuNR damage at the wavelength for the longitudinal plasmon resonance, is adopted to reshape the AuNRs into gold nanospheres. Then, the existence of the AuNRs in designated positions of the fabricated 3D microstructures can be achieved. The doped AuNRs with two-photon luminescence also act as contrast agent for internal diagnosis of 3D polymer microstructures.