Single-emission fluorescence and non-specific cellular organelle-targeting limit the application of carbon dots (CDs) in bio-imaging. Here, we proposed an adjustable strategy for synthesis of four CDs: Tm1-, Tm2-, Tma-, and Tp-CDs. The synthetic scheme was designed with tartaric acid and m-/p-phenylenediamine as precursors and water/acetone as solvent through hydrothermal/solvothermal synthesis and (or) column chromatography isolation. The maximum productivity of the CDs was 56.2 % and the highest fluorescence quantum yield was 47.8 %. These CDs emitted excitation-independent blue, green, and yellow fluorescence, respectively. The red shift of CDs fluorescence was caused by increasing graphitized conjugated sp2 domain and graphitic nitrogen content of carbon core. The hydrophilicity/lipophilicity and protonation ability of CDs determined their cellular uptake, intracellular trafficking and subcellular targets, including endoplasmic reticulum, lysosomes and nucleus. These CDs have the potential application in fluorescence visualization of live bacteria and in the organelle-targeted imaging of living mammalian, fungal, and plant cells. The study provides novel insights into rational design of CDs with multi-color fluorescence and organelle-specific targeting in live cells.