A simple, sensitive and reliable dopamine (DA) biosensor was developed based on a carbon dots (CDs) and chitosan (CS) composite film modified glassy carbon electrode (CDs–CS/GCE). Under optimal conditions, the CDs–CS/GCE showed a better electrochemical response for the detection of DA than that of the glassy carbon electrode (GCE). The oxidation peak current (Ipa) of DA was linear with the concentration of DA in the range from 0.1 μM to 30.0 μM with the limit of detection as 11.2 nM (3S/N). The CDs–CS/GCE was applied to the detection of DA content in an injection solution of DA with satisfactory results.