Carbon-dots (CDs), the emerging fluorescent nanoparticles, show special multicolor properties, chemical stability, and biocompatibility, and are considered as the new and advanced imaging probe in replacement of molecular fluorophores and semiconductor quantum dots. However, the requirement of external high power light source limits the application of fluorescent nanomaterials in bio-imaging. The present study aims to take advantage of bioluminescence resonance energy transfer mechanism (BRET) in creating self-illuminating C-dots. Renilla luciferase (Rluc) is chosen as the BRET donor molecule. Conjugation of Renilla luciferase and C-dots is necessary to keep their distance close for energy transfer. The optimal condition for achieving BRET is investigated by studying the effects of different factors on the performance of BRET, including the type of conjugation, concentration of carbon dots, and conjugation time. The linear relationship of BRET efficiency as a function of the amount of C-dots in the range of 0.20-0.80 mg/mL is observed. The self-illuminating carbon dots could be applied in bioimaging avoiding the tissue damage from the external high power light source.