Fluorescent carbon dots (CDs) with long wavelength emission have attracted increasing attention due to their promising application prospects in biological fields. CDs with long wavelength emission have been synthesized mainly with high temperature and high pressure, while their synthesis at room temperature is relatively rarely studied. In this paper, blue-green luminescent tunable CDs were prepared by alkaline catalysis, and only two steps were required. The fructose and sodium hydroxide solutions were mixed firstly, followed by dialyzing without any additional energy input or external heating. The synthesized CDs were studied by transmission electron microscopy, steady-state fluorescence, and UV-Vis absorption spectroscopy. Moreover, circular dichroic spectrophotometer and picosecond time-correlated single photon counting system was used to analyze the interaction mechanism between CDs and bovine hemoglobin (BHb). Although there have been some studies about the detection of BHb using carbon dots, previous studies mainly focus on the detection of BHb rather than the fluorescence quenching mechanism of carbon dots. Stern-Volmer imagesof the interaction and the influence of BHb on the fluorescence lifetime of CDs have been measured, implying the contribution of static fluorescence quenching. In this process, the content of the α-helical structure of BHb has decreasedby about 3%, demonstrating that the secondary structures of BHb were changed after interacting with CDs. With the addition of BHb, the complexes of BHb and CDs were formed so the fluorescence of CDs decreased. Moreover, the addition of interference samples in the experiment of BHb detection confirmed that the proposed CDs were highly selective, and the variation of reaction times further revealed that the CDs had high stability. It is noticed that, when mixed with BHb, the fluorescence intensity of CDs decreased gradually, and the proportion of decline was linearly related to the concentrations of BHb. Therefore, the biosensors based on CD fluorescence quenching could be established for the specific detection of trace BHb with a linear concentration range from 0 to 5 μmol·L-1 and a detection limit of 243 nmol·L-1 (S/N=3). The CDs were also excited with different wavelengths at 370 and 425 nm, and the results proved that the fluorescence intensities of CDs had a good linear relationship with the concentrations of BHb for both excitation wavelengths, which might provide more application values for the detection of BHb. Due to its convenient synthesis, simple operation and easy availability, the proposed CD probe is of great significance in life sciences and criminal investigation.