In this study, a green and fast method was developed to synthesize high-yield carbon dots (CDs) via one-pot microwave treatment of banana peels without using any other surface passivation agents. Then the as-prepared CDs was used as the reducing agent and stabilizer to synthesize a Pd-Au@CDs nanocomposite by a simple sequential reduction strategy. Finally, Pd-Au@CDs nanocomposite modified glassy carbon electrode (Pd-Au@CDs/GCE) was obtained as a biosensor for target DNA after being immobilized a single-stranded probe DNA by a carboxyl ammonia condensation reaction. Under the optimal conditions, the sensor could detect target DNA concentrations in the range from 5.0×10–16 to 1.0×10−1°molL−1. The detection limit (LD) was estimated to be 1.82×10–17molL−1, which showed higher sensitivity than other electrochemical biosensors reported. In addition, the DNA sensor was also successfully applied to detect colitoxin DNA in human serum. Display omitted •One-step green synthesis of high-yield carbon dots (CDs) from banana peels.•CDs as reductant and stabilizer for synthesizing Pd-Au@CDs composites.•Development of an ultrasensitive electrochemical DNA biosensor based on Pd-Au@CDs composites with a ultralow detection limit of 1.82×10–17molL−1 for colitoxin DNA.