To prepare carbon dots (CDs), there are numerous protocols that use a wide variety of carbon sources, which results in properties of CDs that are unpredictable and highly variable. Therefore, the development of reliable approaches for precisely regulating the nature of CDs is urgently required. Herein, a series of organophilic/hydrophobic CDs (OCDs) were prepared under microwave agitation with ionic liquid 1-alkyl-3-methylimidazolium dicyanamide as the precursor, by varying the alkyl chain linked in the cationic imidazolium moiety. The physicochemical, optical and biological properties, and imaging performance of OCDs were exploited to elucidate the structure–activity relationship, and it was discovered that the alkyl chain plays key roles in governing the properties of OCDs. The increase in the alkyl chain length, from ethyl, butyl, hexyl, and octyl to decyl, led to a remarkable variation in the properties of the OCDs, i.e., a reduction in nitrogen doping from 40.71 to 20.75%, a decrease in binding affinity with bovine serum albumin (BSA), and an increase in cytotoxicity. The interaction of OCDs with BSA and the formation of a ‘protein corona’ substantially increased the biocompatibility of the OCDs. The OCDs penetrated into MCF-7 human breast cancer cells in 10 min and demonstrated bright fluorescence imaging.