In this study, sorted nitrogen-doped graphene quantum dots were prepared and subsequently conjugated with polymers. The synthesized materials exhibited excitationwavelength-independent photoluminescence emissions ranging from ultraviolet to near-infrared and were 0.9−8.4 nm in size. The materials also exhibited high-photoluminescence quantum yields and excellent two-photon properties. Therefore, in twophoton bioimaging the materials with different emission spectra can be effective two-photon contrast agents. Specific antibodies were used to label organelles in cancer cells and identify nuclear antigens, thereby enabling the simultaneous detection of four targets in cells at a single two-photon excitation wavelength. The sorted nitrogen-doped graphene quantum dot materials were determined to be considerably more advantageous than organic dyes in identifying multiplexed targets, and they can be effective probes in cellular imaging.