Here we use natural Chinese paprika to prepare a new kind of amphiphilic carbon dot (A-Dot) that exhibits bright, multicolored fluorescence and contains hydrophilic groups as well as lipophilic capsanthin tails on the surface. It is found that the capsanthin tails in a phospholipid-like structure can promote cell internalization of the A-Dots via crossing cell membranes rapidly in an energy-independent fashion. Compared to highly hydrophilic carbon dots (H-Dots), a control sample prepared from the microwave thermolysis of citric acid and ethylenediamine, our synthesized A-Dots can be taken up by CHO, HeLa, and HFF cells more easily. More importantly, we develop a method to calibrate the hydrophilic lipophilic balance (HLB) values of various kinds of carbon dots (C-Dots). HLB values of A-Dots and H-Dots are determined to be 6.4 and 18.4, respectively. Moreover, we discover that the cellular uptake efficiency of C-Dots is closely related to their HLBs, and the C-Dots with an HLB value of around 6.4 cross the cell membrane easier and faster. As we regulate the HLB value of the A-Dots from 6.4 to 15.3 by removing the capsanthin tails from their surfaces via alkali refluxing, it is found that the refluxed A-Dots can hardly cross HeLa cell membranes. Our work is an essential step toward understanding the importance of regulating the HLB values as well as the surface polarity of the C-Dots for their practical use in bioimaging and also provides a simple but effective way to judge whether the C-Dots in hand are appropriate for cell imaging.