N,P-codoped carbon dots (N,P-CDs) are obtained by a one-pot hydrothermal synthesis strategy using L-arginine and phosphoric acid as the raw materials. The resulting N,P-CDs show good water-solubility and perfect luminescence properties with a high fluorescence quantum yield (18.38%). The maximum emission intensity of CDs is obtained at 444 nm with an excitation of 340 nm. Based on the mechanism of inner filter effect (IFE), CDs are used as an efficient and selective fluorescence sensor for the detection of vitamin B₁₂ (VB₁₂). The linear response of CDs towards VB₁₂ is obviously widened in the range of 1.99–98.6 μM and 98.6–176 μM, and the limit of detection (LOD) is 59 nM. This method is well applied to detect VB₁₂ in two types of vitamin pills and blood serums. In addition, it can be used to bioimaging HeLa cells.