A facile one step hydrothermal synthesis approach was proposed to prepare nitrogen and chlorine co-doped carbon dots using l-ornithine hydrochloride as the sole precursor. The configuration and component of carbon dots were characterized by TEM, XPS, and FTIR. The obtained CDs (Orn-CDs) with a mean diameter of 2.1 nm were well monodispersed in aqueous solutions. The as-prepared CDs exhibited a bright blue fluorescence with a high yield of 60%, good photostability and low cytotoxicity. The emission of Orn-CDs could be selectively and effectively suppressed by Fe3+. Thus, a quantitative assay of Fe3+ was realized by this nanoprobe with a detection limit of 95.6 nmol L-1 in the range of 0.3-50 µmol L-1. Furthermore, ascorbic acid could recover the fluorescence of Orn-CDs suppressed by Fe3+, owing to the transformation of Fe3+ to Fe2+ by ascorbic acid. The limit of detection for ascorbic acid was 137 nmol L-1 in the range of 0.5-10 µmol L-1. In addition, the established method was successfully applied for Fe3+ and ascorbic acid sensing in human serum and urine specimans and for imaging of Fe3+ in living cells. With merits of low economic cost, easy to scale up, without additional functionalized and sample pretreatment, Orn-CDs based sensing platform showed its potential to be used for biomedical related study.