A sensitive, selective, environmentally friendly, high-throughput, well-plate-based immunosorbent assay was developed to detect human α-fetoprotein (AFP) using carbon dots (C-Dots). Highly fluorescent C-Dots were synthesized using a one-step hydrothermal reaction, with citric acid serving as the carbon source and ethylene diamine acting as the nitrogen source. The reaction conditions were optimized to obtain the desired surface functionality. Then, the C-Dots were used to label one member of the anti-AFP pair (Ab2) via amine-amine coupling using glutaraldehyde. The capture anti-AFP (Ab1) was coated onto polystyrene well plates and bovine serum albumin (BSA) was used to block unsaturated binding sites. AFP was incubated in Ab1-coated wells; unbound AFP was then washed away with Tween-20. Next, the C-Dot-labeled Ab2 was added to form a sandwich immunocomplex with the AFP bound to the Ab1-coated wells. The fluorescence intensities detected from the C-Dots on these sandwich immunocomplexes were positively correlated to the concentrations of AFP antigen. A five-parameter logistic regression calibration curve was established between fluorescence and clinically important AFP concentrations (range: 0-350 ng/mL with a correlation coefficient of R(2) = 0.995). The results from the C-Dot-based immunoassay were in agreement with results from traditional immunoassays, which used horseradish peroxidase (HRP, R(2) = 0.964) and fluorescein isothiocyanate (FITC, R(2) = 0.973). These results indicated that C-Dots have great potential to be applied as biolabels for high-throughput well-plate-based immunoassays.