Vitamins are indispensable active components of the human body, and their abnormal levels are often closely related to various diseases. Therefore, searching for ultra-sensitive and accurate vitamin detection strategies is essential and significant for clinical examination and diagnosis. In this paper, multi-wavelength emission fluorescent nitrogen-doped carbon dots (N-doped CDs) were synthesized by a one-step solvothermal method using four-leaf green fluorescent extract, O-phenylenediamine, and β-cyclodextrin as the raw materials, and N, N-dimethylformamide as the solvent. The CDs can achieve controllable fluorescence changes with multi-wavelength emission at different excitation wavelengths and exhibit good photoluminescence optical properties. In addition, the fluorescence emission peak intensities of the N-doped CDs at 400, and 470 nm excitation wavelengths decreased with increased AA content. They showed good linearity in the 0.6-1.4, and 0.1-0.5 μM ranges, with detection limit coefficients of 0.12, and 0.14 μM, respectively. Moreover, due to their excellent photostability and good biocompatibility, N-doped CDs have been successfully applied to the bioimaging and biosensing of exogenous AA in 4T1 cells, as well as in vivo imaging tracking of Kunming (KM) mice.