We report on an ultrasensitive and selective fluorescence assay for Sudan I and III against the influence of Sudan II and IV based on ligand exchange mechanism. Calcein as a fluorescence indicator and Sudan I-IV as model analytes were employed to investigate the analytical feature of this assay platform. Results show that the fluorescence of calcein can be efficiently quenched by Cu(II). When the ligand exchange reaction proceeds, calcein is deprived of Cu(II) by Sudan I and III, resulting in the fluorescence recovery of calcein. However, the ligand exchange reaction does not happen in the presence of Sudan II or IV due to the 2-methyl steric effects, which is favorable for selective determination of Sudan I and III against the influence of Sudan II and IV. It was found that the fluorescence enhancement efficiency (FEE) against the concentration of Sudan (c(Sudan), nmol L⁻¹) shows a linear relationship. The calibration equations are FEE(Sudan I)=0.0032 c(Sudan I)-0.02613, and FEE(Sudan III)=0.0033 c(Sudan III)-0.02467 over the corresponding linear range of 11.25-2078.29 and 9.44-1035.78 nmol L⁻¹ with the correlation coefficients (R(2)) of 0.9984 and 0.9955, respectively. And the detection limits (3σ/slope) are calculated to be 211.3 and 208.5 pmol L⁻¹ for Sudan I and III, respectively, showing ultralow detection limit. The Sudan dye in a commercial chilli powder sample was assayed with satisfactory results.