<p>The demand of fluorometric detection on Fe³⁺ ions and hemoglobin are highly increased in environmental detection, anemia diagnosis, and criminal investigation. To enhance the performance of fluorometric detection of mentioned analytes, good water solubility, ease of synthesis, low cost, and high quantum yields are highly required. In this paper, the morphological, structural, luminescent characteristics of CDs, and their fluorometric detection of Fe³⁺ and hemoglobin were analyzed. The carbon dots (CDs) based on o-toluidine were synthesized via hydrothermal method. The CDs exhibit a two-dimensional crystallite structure with good hydrophilic properties. Also, the CDs possess blue dominant luminescence under 240 nm light excitation. The blue luminescence was reduced by increasing Fe³⁺ and hemoglobin detection. Compared with other heavy metal ions, the CDs-Fe³⁺ system showed high luminescence reduction. The limit of detection of Fe³⁺ and hemoglobin were calculated to be 0.133 μM and 72 nM, respectively. Comparison of luminescence decay curve, overlap of analyte absorption and luminescence spectra show that the reduction of luminescence intensity is due to FRET mechanism of CDs-Fe³⁺ ions and -hemoglobin. These results indicate that the CDs can be applied as a luminescent material in fluorometric detection for environmental detection, anemia diagnosis, and criminal investigation.</p>