Fluorescein (FL) has been proven to be a very effective fluorescent tracer in pharmaceutical and medical applications owing to its high quantum yield of fluorescence in physiological conditions and its high membrane permeability. In order to protect FL from metabolic effects during its journey in the blood stream, proteins such as human serum albumin (HSA) were used as carriers. In this way, the monoanion and the dianion forms of FL, the only fluorescent species, can be preserved. In the present work, we used several cyclodextrins (CDs) as macromolecular hosts of FL. We characterized the spectroscopic behavior of FL in different CDs and determined the stoichiometric ratios and the binding constants of their complexes. Using FL to probe the CDs reveals the degree of hydrophobicity of the cavities. As the cavity size decreases in the order y-CD to B-CD to a-CD, the probe molecule has more encapsulation effect, which is shown as a slight increase in the monoanion signal in the absorption and fluorescence spectra, relative to those of the dianion. The measured fluorescence lifetimes show the same trend. The spectral changes and the measured fluorescence lifetimes of FL with B-CD and its derivatives indicate that the largest encapsulation effect is due to the FL/DMB-CD (2,6-di-Omethyl-B-CD) complex. This observation is correlated to the more hydrophobic cavity of DMB-CD which is due to the methylation of two hydroxyl groups in the parent BCD. On the contrary, less encapsulation was observed in the FL/TMB-CD (2,3,6-tri-Omethyl-B-CD) complex which is due to a steric effect at the secondary rim of the CD that prevents a full encapsulation of the FL guest. The stoichiometry of the complexes was dominated by 1:1 ratio, except in FL/TMB-CD where the stoichiometry was possible up to 1:2 (one FL and two CDs). The latter observation can be due to the incomplete encapsulation of the guest molecule in which the FL molecule may interact with the rim part of two TMB-CD molecules in order to stabilize the complex formation. The estimated binding (association) constants indicate large values for the complex formation of FL with a-CD and DMB-CD, in agreement with the observed large hydrophobic effect in both complexes. Varying the pH of the FL aqueous solution in the range 2-13 allowed the estimation of the different pKa values of the different ionic and protonated species. From the absorbance change, the following pKa values were calculated; 2.30 (protonated FL), 4.30 (monoanion FL), and 5.90 (dianion FL). In the excited state, the dianion species dominates over a wide range of pH, and the estimated pKq* was 5.90. Fluorescence lifetime measurements in the same pH range confirmed the dominant signal of the dianion form of FL over a wide pH range. Two fluorescence lifetimes were measured and were assigned to the monoanion (~1.8 ns) and the dianion (~ 4.5 ns) species. The current results suggest the possible use of some CDs as carriers for FL, instead of using proteins. One such application is encapsulating FL in a proper CD when studying its penetration through the blood-ocular barriers during certain eye exams. In this regard, the metabolic effect on FL, which depletes its fluorescence intensity, can be minimized during the exam period if FL is trapped inside a CD cavity. Our results indicate that FL has a stronger binding with a-CD and DMB-CD, than its binding with HSA (often used for such eye tests), which may offer a better delivery tool for FL to its target. In addition, using CDs is much cheaper than using HSA as a carrier. CDs are also compatible with the blood components and are not expected to cause any harm when used as carriers.