In recent years, fluorescent nanomaterials have attracted great attention due to their broad applications. Extensive efforts have been made on the development of novel metal-free fluorescent nanomaterials. Among these nanomaterials, graphene quantum dots (GQDs) have demonstrated chemical inertness, photostability, low cytotoxicity and favorable biocompatibility. Due to their excellent quantum confinement effect and edge effect, GQDs are usually characterized with distinct photoluminescence (PL) behaviors for biomedical imaging. Similar to GQDs, graphitic-phase C₃N₄ (g-C₃N₄) nanosheets have also exhibited high biocompatibility, low toxicity, and unique optical properties. Both GQDs and g-C₃N₄ nanosheets have been extensively investigated as potential biomedical imaging candidates. However, there are very few systematic comparative studies on GQDs and g-C₃N₄ nanosheets in terms of photoluminescence properties and biological imaging characteristics. Therefore, in this study, we mainly focus on investigating these differences. Highly water-dispersible GQDs and g-C₃N₄ nanosheets were prepared by a similar top-down process, chemically oxidizing thermally exfoliated graphite oxide and bulk g-C₃N₄ with concentrated nitric acid, respectively. UV-Vis absorption and photoluminescence spectral analyses were used to compare their optical properties. Compared to GQDs, g-C₃N₄ nanosheets demonstrate higher photoluminescence quantum yield. Cytotoxicity measurements indicate that both GQDs and g-C₃N₄ nanosheets are of low cytotoxicity with favorable biocompatibility. The remarkable performances of GQDs and g-C₃N₄ nanosheets in biomedical imaging are demonstrated by a simple incubation study with HepG2 cells. All these studies suggest that both GQDs and g-C₃N₄ nanosheets prepared by the above method have low cytotoxicity and are greatly promising for biomedical applications, such as in vitro and in vivo imaging studies.