Carbon dots (CDs) are attracting significant research attention due to their low toxicity and highly favorable photochemical properties. This study reports the synthesis of nitrogen‐doped CDs (N‐CDs) using 3‐aminophenol and o‐phenylenediamine as carbon precursors and their application to nontoxic, biocompatible polychromatic agents for biological imaging studies. The prepared N‐CDs exhibit multicolored fluorescence (blue, green, yellow, orange) with excellent optical stability and biocompatibility. As a result of Fourier‐transform infrared spectroscopy and X‐ray photoelectron spectroscopy, N‐CD is found to have similar –OH and C–H in the surface functional group, showing a graphene structure with good hydrophilicity. The size of the synthesized N‐CDs is confirmed by transmission electron microscopy images to be less than 10 nm. The 3‐4,5‐dimethylthiazol‐2,5‐diphenyl tetrazolium bromide analysis is performed using HeLa cells, and cell survival is very high in all N‐CDs. Among N‐CDs, G‐CDs exhibit the highest fluorescence intensity and provide excellent fluorescence microscopic images for cells in blue, green, and red channels.