Carbon dots (C-dots) with multicolour fluorescence feature are presently gaining much importance in the area of bioanalytics and bioimaging due to their biocompatible and strong fluorescent nature. Herein, we report a one-step method for synthesizing multicolour, surface passivated carbon dots using chitosan as a 'greener' source through microwave pyrolysis approach. The present study also deals with the synthesis of chitosan-pDNA (GFP) nanocomplexes for gene delivery applications. Development of such biofriendly imaging agents and nanocarriers has huge diagnostic and therapeutic potential.