In this work, we develop a facile one-step strategy for rapidly (within 1 min) preparing four kinds of nitrogen doped carbon dots (N-CDs) and construct a label free sensor array for protein discrimination. The monodispersed blue emitting N-CDs, with a quantum yield (QY) of about 7.6% (calibrated with quinine sulfate as the reference), were systematically characterized by using fluorescence spectroscopy, atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR). Based on the diverse fluorescence intensity response patterns, eight proteins with diverse isoelectric points and molecular weights could be simultaneously and effectively discriminated at nanomolar concentration by linear discrimination analysis (LDA). Proteins with different concentrations or protein mixtures can also be well identified. Furthermore, multiple proteins could still be well identified without any overlap in the presence of human urine. Moreover, serums from rectal cancer patients, Alzheimer's disease patients and healthy people could be effectively discriminated, showing great potential in auxiliary clinical diagnosis.