The dynamics of the nucleolus in living cells are important in understanding cellular behavior and malignant transformation. However, construction of a fluorescent probe for long-term monitoring of nucleolus is significantly constrained by the cost, cellular barriers, cytotoxicity, and photobleaching. Herein, a one-pot microwave-assisted synthesis is developed for facile and quick preparation (domestic microwave oven, 7.5 min) of fluorescent carbon dots (CDs) for nucleolus imaging and tracking merely by adding protamine (PTM) into the precursor solution. The as-prepared carbon dots (CD-PTMs) feature photostability, biocompatibility, multicolor emission, and fluorescence enhancement upon binding with RNA. Significantly, CD-PTMs exhibit the capability of crossing both the cytoplasm and nuclear membranes, and located at the nucleolus for long-term (at least 12 h) imaging studies. Moreover, CD-PTMs are demonstrated to be capable of serving as efficient nanocarriers for in vitro transfection applications by taking advantage of the positively charged surface PTM derivative, showing the potential of CD-PTMs as multifunctional nanoprobes for the bioapplications.