The nucleolus is an important subnuclear structure in the cell, which plays an important role in the evolution of malignancies and the diagnosis of cancer. Although the nucleolus is critical, so far, there are really few fluorescent probes for the nucleolus. In this paper, salicylic acid and 1,8-diaminonaphthalene were used as carbon and nitrogen sources, and a novel fluorescent carbon nanoparticles (FCNs) were synthesized by microwave digestion system. Its physical, chemical and optical properties of FCNs were characterized and analyzed by using transmission electron microscope (TEM), dynamic light scattering instrument (DLS), Fourier infrared spectrometer, ultraviolet spectrophotometer, fluorescence spectrometer, etc. The experimental results demonstrated that the synthesized carbon nanoparticles were uniform in size and rich with amino groups on surface. Its best excitation wavelength was 348 nm, the corresponding maximum emission peak was 432 nm. Its fluorescence quantum yield was 17.8% and fluorescence lifetime was 1.13 ns. Moreover, the photostability and cytotoxicity of FCNs were investigated with illumination and MTT assays. Our results suggest that FCNs exhibit to be a really photostable and low toxic probe. To confirm the potential application of this fluorescence FCNs in bioimaging, the FCNs were designed to stain human cervical cancer HeLa cells. After incubation, we found that FCNs enable the function of selectively staining the nucleolus of living cells due to its positive chargeability. Its cellular uptake mechanism and intracellular distribution in HeLa cells were explored further by using confocal laser scanning microscopy and TEM. It was interesting to observe that more FCNs entered the cell, and lighted the nucleolus more obviously with the extension of the co-incubation time. In addition, after investigating the cells’ uptake path of FCNs, we found that FCNs were endocytosed via caveolae-mediated pathway rather than clathrin mediated pathways or macropinocytosis. Our study provided a potential probe for nucleolus and this finding is of great significance for the development of functional nanomaterials targeting subcellular organelles.